EP4085150A1 - Genome-scale imaging of the 3d organization and transcriptional activity of chromatin - Google Patents
Genome-scale imaging of the 3d organization and transcriptional activity of chromatinInfo
- Publication number
- EP4085150A1 EP4085150A1 EP20910372.0A EP20910372A EP4085150A1 EP 4085150 A1 EP4085150 A1 EP 4085150A1 EP 20910372 A EP20910372 A EP 20910372A EP 4085150 A1 EP4085150 A1 EP 4085150A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- nucleic acid
- sample
- determining
- readout
- probes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000003384 imaging method Methods 0.000 title claims abstract description 490
- 108010077544 Chromatin Proteins 0.000 title claims abstract description 401
- 210000003483 chromatin Anatomy 0.000 title claims abstract description 401
- 230000008520 organization Effects 0.000 title claims abstract description 98
- 230000002103 transcriptional effect Effects 0.000 title claims abstract description 69
- 238000000034 method Methods 0.000 claims abstract description 412
- 210000000349 chromosome Anatomy 0.000 claims abstract description 199
- 230000035897 transcription Effects 0.000 claims abstract description 58
- 238000013518 transcription Methods 0.000 claims abstract description 58
- 239000000523 sample Substances 0.000 claims description 641
- 210000004027 cell Anatomy 0.000 claims description 453
- 108020004711 Nucleic Acid Probes Proteins 0.000 claims description 307
- 239000002853 nucleic acid probe Substances 0.000 claims description 307
- 102000039446 nucleic acids Human genes 0.000 claims description 302
- 108020004707 nucleic acids Proteins 0.000 claims description 302
- 150000007523 nucleic acids Chemical class 0.000 claims description 302
- 230000011664 signaling Effects 0.000 claims description 274
- 210000004940 nucleus Anatomy 0.000 claims description 177
- 108090000623 proteins and genes Proteins 0.000 claims description 148
- 239000002773 nucleotide Substances 0.000 claims description 85
- 125000003729 nucleotide group Chemical group 0.000 claims description 83
- 230000027455 binding Effects 0.000 claims description 76
- 230000000295 complement effect Effects 0.000 claims description 63
- 102000004169 proteins and genes Human genes 0.000 claims description 43
- 210000002353 nuclear lamina Anatomy 0.000 claims description 42
- 230000000415 inactivating effect Effects 0.000 claims description 38
- 238000001514 detection method Methods 0.000 claims description 36
- 238000004422 calculation algorithm Methods 0.000 claims description 33
- 238000012937 correction Methods 0.000 claims description 23
- 238000000386 microscopy Methods 0.000 claims description 22
- 230000009870 specific binding Effects 0.000 claims description 20
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 15
- 238000000799 fluorescence microscopy Methods 0.000 claims description 9
- 230000000903 blocking effect Effects 0.000 claims description 8
- 238000004061 bleaching Methods 0.000 claims description 6
- 230000015556 catabolic process Effects 0.000 claims description 6
- 238000006731 degradation reaction Methods 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 230000003287 optical effect Effects 0.000 claims description 5
- 238000007476 Maximum Likelihood Methods 0.000 claims description 2
- 239000002105 nanoparticle Substances 0.000 claims description 2
- 239000000376 reactant Substances 0.000 claims description 2
- 238000012634 optical imaging Methods 0.000 claims 1
- 230000003993 interaction Effects 0.000 abstract description 139
- 238000007901 in situ hybridization Methods 0.000 abstract description 5
- 238000013507 mapping Methods 0.000 abstract description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 217
- 108020004414 DNA Proteins 0.000 description 141
- 238000009396 hybridization Methods 0.000 description 139
- 238000009826 distribution Methods 0.000 description 85
- 230000000875 corresponding effect Effects 0.000 description 64
- 238000002474 experimental method Methods 0.000 description 59
- 239000011159 matrix material Substances 0.000 description 54
- 238000013459 approach Methods 0.000 description 48
- 239000000872 buffer Substances 0.000 description 48
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 40
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 36
- 238000004458 analytical method Methods 0.000 description 33
- 235000018102 proteins Nutrition 0.000 description 30
- 238000006073 displacement reaction Methods 0.000 description 29
- 238000005286 illumination Methods 0.000 description 29
- 238000005259 measurement Methods 0.000 description 29
- 238000009413 insulation Methods 0.000 description 28
- 230000008859 change Effects 0.000 description 27
- 108091034117 Oligonucleotide Proteins 0.000 description 26
- 238000005204 segregation Methods 0.000 description 26
- 230000006870 function Effects 0.000 description 24
- 230000000670 limiting effect Effects 0.000 description 22
- 238000010304 firing Methods 0.000 description 21
- 230000004807 localization Effects 0.000 description 21
- 108010014064 CCCTC-Binding Factor Proteins 0.000 description 20
- 102000016897 CCCTC-Binding Factor Human genes 0.000 description 20
- 101800002638 Alpha-amanitin Proteins 0.000 description 18
- RXGJTYFDKOHJHK-UHFFFAOYSA-N S-deoxo-amaninamide Natural products CCC(C)C1NC(=O)CNC(=O)C2Cc3c(SCC(NC(=O)CNC1=O)C(=O)NC(CC(=O)N)C(=O)N4CC(O)CC4C(=O)NC(C(C)C(O)CO)C(=O)N2)[nH]c5ccccc35 RXGJTYFDKOHJHK-UHFFFAOYSA-N 0.000 description 18
- 239000004007 alpha amanitin Substances 0.000 description 18
- CIORWBWIBBPXCG-SXZCQOKQSA-N alpha-amanitin Chemical compound O=C1N[C@@H](CC(N)=O)C(=O)N2C[C@H](O)C[C@H]2C(=O)N[C@@H]([C@@H](C)[C@@H](O)CO)C(=O)N[C@@H](C2)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@H]1C[S@@](=O)C1=C2C2=CC=C(O)C=C2N1 CIORWBWIBBPXCG-SXZCQOKQSA-N 0.000 description 18
- CIORWBWIBBPXCG-UHFFFAOYSA-N alpha-amanitin Natural products O=C1NC(CC(N)=O)C(=O)N2CC(O)CC2C(=O)NC(C(C)C(O)CO)C(=O)NC(C2)C(=O)NCC(=O)NC(C(C)CC)C(=O)NCC(=O)NC1CS(=O)C1=C2C2=CC=C(O)C=C2N1 CIORWBWIBBPXCG-UHFFFAOYSA-N 0.000 description 18
- 229960005502 α-amanitin Drugs 0.000 description 18
- 108700009124 Transcription Initiation Site Proteins 0.000 description 17
- 239000000975 dye Substances 0.000 description 17
- 238000009877 rendering Methods 0.000 description 17
- 239000000243 solution Substances 0.000 description 17
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 16
- 239000011324 bead Substances 0.000 description 16
- 238000013442 quality metrics Methods 0.000 description 16
- 239000012530 fluid Substances 0.000 description 14
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 13
- 230000003321 amplification Effects 0.000 description 13
- 230000015572 biosynthetic process Effects 0.000 description 13
- 230000002759 chromosomal effect Effects 0.000 description 13
- 238000003199 nucleic acid amplification method Methods 0.000 description 13
- 239000002751 oligonucleotide probe Substances 0.000 description 13
- 230000002441 reversible effect Effects 0.000 description 13
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 12
- -1 IncRNA Proteins 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 238000002372 labelling Methods 0.000 description 12
- 230000005758 transcription activity Effects 0.000 description 12
- 239000003086 colorant Substances 0.000 description 11
- 238000013461 design Methods 0.000 description 11
- 230000000694 effects Effects 0.000 description 11
- 238000010166 immunofluorescence Methods 0.000 description 11
- 229930040373 Paraformaldehyde Natural products 0.000 description 10
- 229920002866 paraformaldehyde Polymers 0.000 description 10
- 230000008685 targeting Effects 0.000 description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 230000004075 alteration Effects 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 238000010790 dilution Methods 0.000 description 9
- 239000012895 dilution Substances 0.000 description 9
- 230000005764 inhibitory process Effects 0.000 description 9
- 230000014632 RNA localization Effects 0.000 description 8
- GLEVLJDDWXEYCO-UHFFFAOYSA-N Trolox Chemical compound O1C(C)(C(O)=O)CCC2=C1C(C)=C(C)C(O)=C2C GLEVLJDDWXEYCO-UHFFFAOYSA-N 0.000 description 8
- 230000002596 correlated effect Effects 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 108091092195 Intron Proteins 0.000 description 7
- 102000006382 Ribonucleases Human genes 0.000 description 7
- 108010083644 Ribonucleases Proteins 0.000 description 7
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 7
- 238000012512 characterization method Methods 0.000 description 7
- 238000004891 communication Methods 0.000 description 7
- 230000005284 excitation Effects 0.000 description 7
- 230000014509 gene expression Effects 0.000 description 7
- XXMIOPMDWAUFGU-UHFFFAOYSA-N hexane-1,6-diol Chemical compound OCCCCCCO XXMIOPMDWAUFGU-UHFFFAOYSA-N 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 230000002829 reductive effect Effects 0.000 description 7
- 239000011534 wash buffer Substances 0.000 description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- 238000004113 cell culture Methods 0.000 description 6
- 238000010276 construction Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 238000010191 image analysis Methods 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 230000037452 priming Effects 0.000 description 6
- 230000003252 repetitive effect Effects 0.000 description 6
- 230000000717 retained effect Effects 0.000 description 6
- 238000010839 reverse transcription Methods 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 241000283074 Equus asinus Species 0.000 description 5
- 102000007999 Nuclear Proteins Human genes 0.000 description 5
- 108010089610 Nuclear Proteins Proteins 0.000 description 5
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 5
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 5
- 108091093037 Peptide nucleic acid Proteins 0.000 description 5
- 230000022131 cell cycle Effects 0.000 description 5
- 239000003623 enhancer Substances 0.000 description 5
- 108020004999 messenger RNA Proteins 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 108020004418 ribosomal RNA Proteins 0.000 description 5
- 230000011218 segmentation Effects 0.000 description 5
- 238000012800 visualization Methods 0.000 description 5
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 4
- 102000004064 Geminin Human genes 0.000 description 4
- 108090000577 Geminin Proteins 0.000 description 4
- 108010015776 Glucose oxidase Proteins 0.000 description 4
- 239000004366 Glucose oxidase Substances 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 229920001213 Polysorbate 20 Polymers 0.000 description 4
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 210000002230 centromere Anatomy 0.000 description 4
- 108010045512 cohesins Proteins 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 230000001973 epigenetic effect Effects 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- 239000012737 fresh medium Substances 0.000 description 4
- 230000005714 functional activity Effects 0.000 description 4
- 229940116332 glucose oxidase Drugs 0.000 description 4
- 235000019420 glucose oxidase Nutrition 0.000 description 4
- 238000003505 heat denaturation Methods 0.000 description 4
- 238000007654 immersion Methods 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 229920002113 octoxynol Polymers 0.000 description 4
- 239000003921 oil Substances 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 238000001353 Chip-sequencing Methods 0.000 description 3
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 3
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 102000015097 RNA Splicing Factors Human genes 0.000 description 3
- 210000001766 X chromosome Anatomy 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 239000003638 chemical reducing agent Substances 0.000 description 3
- 230000001276 controlling effect Effects 0.000 description 3
- 230000004049 epigenetic modification Effects 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 238000011835 investigation Methods 0.000 description 3
- 238000002844 melting Methods 0.000 description 3
- 230000008018 melting Effects 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 230000002572 peristaltic effect Effects 0.000 description 3
- 238000000513 principal component analysis Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 230000009897 systematic effect Effects 0.000 description 3
- 230000007704 transition Effects 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- JLDSMZIBHYTPPR-UHFFFAOYSA-N Alexa Fluor 405 Chemical compound CC[NH+](CC)CC.CC[NH+](CC)CC.CC[NH+](CC)CC.C12=C3C=4C=CC2=C(S([O-])(=O)=O)C=C(S([O-])(=O)=O)C1=CC=C3C(S(=O)(=O)[O-])=CC=4OCC(=O)N(CC1)CCC1C(=O)ON1C(=O)CCC1=O JLDSMZIBHYTPPR-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 108090001008 Avidin Proteins 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 102100035882 Catalase Human genes 0.000 description 2
- 108010053835 Catalase Proteins 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 230000008836 DNA modification Effects 0.000 description 2
- 230000004543 DNA replication Effects 0.000 description 2
- 102100029952 Double-strand-break repair protein rad21 homolog Human genes 0.000 description 2
- 101710185850 Exodeoxyribonuclease Proteins 0.000 description 2
- 108010002700 Exoribonucleases Proteins 0.000 description 2
- 102000004678 Exoribonucleases Human genes 0.000 description 2
- 102000016359 Fibronectins Human genes 0.000 description 2
- 108010067306 Fibronectins Proteins 0.000 description 2
- 230000010337 G2 phase Effects 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 108010034791 Heterochromatin Proteins 0.000 description 2
- 102100039869 Histone H2B type F-S Human genes 0.000 description 2
- 108010033040 Histones Proteins 0.000 description 2
- 102000006947 Histones Human genes 0.000 description 2
- 101000584942 Homo sapiens Double-strand-break repair protein rad21 homolog Proteins 0.000 description 2
- 101001035372 Homo sapiens Histone H2B type F-S Proteins 0.000 description 2
- 101000587430 Homo sapiens Serine/arginine-rich splicing factor 2 Proteins 0.000 description 2
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 2
- 101100384865 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) cot-1 gene Proteins 0.000 description 2
- 108091005461 Nucleic proteins Proteins 0.000 description 2
- 229940123973 Oxygen scavenger Drugs 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 108020004518 RNA Probes Proteins 0.000 description 2
- 108010039259 RNA Splicing Factors Proteins 0.000 description 2
- 239000003391 RNA probe Substances 0.000 description 2
- 238000012952 Resampling Methods 0.000 description 2
- 108010057163 Ribonuclease III Proteins 0.000 description 2
- 102000003661 Ribonuclease III Human genes 0.000 description 2
- 230000018199 S phase Effects 0.000 description 2
- 238000010870 STED microscopy Methods 0.000 description 2
- 102100029666 Serine/arginine-rich splicing factor 2 Human genes 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- 108020004459 Small interfering RNA Proteins 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 108020004566 Transfer RNA Proteins 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 238000005904 alkaline hydrolysis reaction Methods 0.000 description 2
- 230000000712 assembly Effects 0.000 description 2
- 238000000429 assembly Methods 0.000 description 2
- 230000002902 bimodal effect Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 230000001427 coherent effect Effects 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 2
- 238000013527 convolutional neural network Methods 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 108010002712 deoxyribonuclease II Proteins 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 229960000633 dextran sulfate Drugs 0.000 description 2
- 210000001840 diploid cell Anatomy 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000001317 epifluorescence microscopy Methods 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 2
- 230000001747 exhibiting effect Effects 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 210000004458 heterochromatin Anatomy 0.000 description 2
- 238000012165 high-throughput sequencing Methods 0.000 description 2
- 210000003917 human chromosome Anatomy 0.000 description 2
- 238000010569 immunofluorescence imaging Methods 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 238000003064 k means clustering Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 239000002480 mineral oil Substances 0.000 description 2
- 235000010446 mineral oil Nutrition 0.000 description 2
- 238000004651 near-field scanning optical microscopy Methods 0.000 description 2
- 108091027963 non-coding RNA Proteins 0.000 description 2
- 102000042567 non-coding RNA Human genes 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 230000003094 perturbing effect Effects 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 2
- 238000010377 protein imaging Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000003161 ribonuclease inhibitor Substances 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 2
- 238000003530 single readout Methods 0.000 description 2
- 239000012279 sodium borohydride Substances 0.000 description 2
- 229910000033 sodium borohydride Inorganic materials 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 2
- 238000011895 specific detection Methods 0.000 description 2
- 239000010902 straw Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 102000055501 telomere Human genes 0.000 description 2
- 108091035539 telomere Proteins 0.000 description 2
- 210000003411 telomere Anatomy 0.000 description 2
- RLNWRDKVJSXXPP-UHFFFAOYSA-N tert-butyl 2-[(2-bromoanilino)methyl]piperidine-1-carboxylate Chemical compound CC(C)(C)OC(=O)N1CCCCC1CNC1=CC=CC=C1Br RLNWRDKVJSXXPP-UHFFFAOYSA-N 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- YWXCBLUCVBSYNJ-UHFFFAOYSA-N 2-(2-sulfanylethylsulfonyl)ethanethiol Chemical compound SCCS(=O)(=O)CCS YWXCBLUCVBSYNJ-UHFFFAOYSA-N 0.000 description 1
- 108010000834 2-5A-dependent ribonuclease Proteins 0.000 description 1
- 102100027962 2-5A-dependent ribonuclease Human genes 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- NOIRDLRUNWIUMX-UHFFFAOYSA-N 2-amino-3,7-dihydropurin-6-one;6-amino-1h-pyrimidin-2-one Chemical compound NC=1C=CNC(=O)N=1.O=C1NC(N)=NC2=C1NC=N2 NOIRDLRUNWIUMX-UHFFFAOYSA-N 0.000 description 1
- QSECPQCFCWVBKM-UHFFFAOYSA-N 2-iodoethanol Chemical compound OCCI QSECPQCFCWVBKM-UHFFFAOYSA-N 0.000 description 1
- SMBSZJBWYCGCJP-UHFFFAOYSA-N 3-(diethylamino)chromen-2-one Chemical compound C1=CC=C2OC(=O)C(N(CC)CC)=CC2=C1 SMBSZJBWYCGCJP-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- SKZQZGSPYYHTQG-UHFFFAOYSA-N 4-[2-[4-(3-phenylpyrazolo[1,5-a]pyrimidin-6-yl)phenoxy]ethyl]morpholine Chemical compound C=1C=C(C2=CN3N=CC(=C3N=C2)C=2C=CC=CC=2)C=CC=1OCCN1CCOCC1 SKZQZGSPYYHTQG-UHFFFAOYSA-N 0.000 description 1
- LELMRLNNAOPAPI-UFLZEWODSA-N 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid;aminophosphonous acid Chemical compound NP(O)O.N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 LELMRLNNAOPAPI-UFLZEWODSA-N 0.000 description 1
- FFKUHGONCHRHPE-UHFFFAOYSA-N 5-methyl-1h-pyrimidine-2,4-dione;7h-purin-6-amine Chemical compound CC1=CNC(=O)NC1=O.NC1=NC=NC2=C1NC=N2 FFKUHGONCHRHPE-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 239000012103 Alexa Fluor 488 Substances 0.000 description 1
- 239000012114 Alexa Fluor 647 Substances 0.000 description 1
- 239000012116 Alexa Fluor 680 Substances 0.000 description 1
- 239000012117 Alexa Fluor 700 Substances 0.000 description 1
- 239000012118 Alexa Fluor 750 Substances 0.000 description 1
- 239000012119 Alexa Fluor 790 Substances 0.000 description 1
- 239000012099 Alexa Fluor family Substances 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- 108091005625 BRD4 Proteins 0.000 description 1
- 102100029892 Bromodomain and WD repeat-containing protein 1 Human genes 0.000 description 1
- 102100029895 Bromodomain-containing protein 4 Human genes 0.000 description 1
- 101001107784 Caenorhabditis elegans Deoxyribonuclease-2 Proteins 0.000 description 1
- 238000010196 ChIP-seq analysis Methods 0.000 description 1
- 108020004998 Chloroplast DNA Proteins 0.000 description 1
- 102000002494 Endoribonucleases Human genes 0.000 description 1
- 108010093099 Endoribonucleases Proteins 0.000 description 1
- 108091007413 Extracellular RNA Proteins 0.000 description 1
- 102100026121 Flap endonuclease 1 Human genes 0.000 description 1
- 230000010190 G1 phase Effects 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 102000017278 Glutaredoxin Human genes 0.000 description 1
- 108050005205 Glutaredoxin Proteins 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 102100021519 Hemoglobin subunit beta Human genes 0.000 description 1
- 108091005904 Hemoglobin subunit beta Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101000794040 Homo sapiens Bromodomain and WD repeat-containing protein 1 Proteins 0.000 description 1
- 101000913035 Homo sapiens Flap endonuclease 1 Proteins 0.000 description 1
- 101001066878 Homo sapiens Polyribonucleotide nucleotidyltransferase 1, mitochondrial Proteins 0.000 description 1
- 101000716740 Homo sapiens SR-related and CTD-associated factor 4 Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 101710203526 Integrase Proteins 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 102000006835 Lamins Human genes 0.000 description 1
- 108010047294 Lamins Proteins 0.000 description 1
- 108020005196 Mitochondrial DNA Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091007412 Piwi-interacting RNA Proteins 0.000 description 1
- 102100034410 Polyribonucleotide nucleotidyltransferase 1, mitochondrial Human genes 0.000 description 1
- 102000009572 RNA Polymerase II Human genes 0.000 description 1
- 108010009460 RNA Polymerase II Proteins 0.000 description 1
- 108090000621 Ribonuclease P Proteins 0.000 description 1
- 102000004167 Ribonuclease P Human genes 0.000 description 1
- 108090000638 Ribonuclease R Proteins 0.000 description 1
- 102100020878 SR-related and CTD-associated factor 4 Human genes 0.000 description 1
- 108020004688 Small Nuclear RNA Proteins 0.000 description 1
- 102000039471 Small Nuclear RNA Human genes 0.000 description 1
- 108020003224 Small Nucleolar RNA Proteins 0.000 description 1
- 102000042773 Small Nucleolar RNA Human genes 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 102100036407 Thioredoxin Human genes 0.000 description 1
- QHNORJFCVHUPNH-UHFFFAOYSA-L To-Pro-3 Chemical compound [I-].[I-].S1C2=CC=CC=C2[N+](C)=C1C=CC=C1C2=CC=CC=C2N(CCC[N+](C)(C)C)C=C1 QHNORJFCVHUPNH-UHFFFAOYSA-L 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000018690 Trypsinogen Human genes 0.000 description 1
- 108010027252 Trypsinogen Proteins 0.000 description 1
- 210000002593 Y chromosome Anatomy 0.000 description 1
- ZVUUXEGAYWQURQ-UHFFFAOYSA-L Yo-Pro-3 Chemical compound [I-].[I-].O1C2=CC=CC=C2[N+](C)=C1C=CC=C1C2=CC=CC=C2N(CCC[N+](C)(C)C)C=C1 ZVUUXEGAYWQURQ-UHFFFAOYSA-L 0.000 description 1
- JSBNEYNPYQFYNM-UHFFFAOYSA-J YoYo-3 Chemical compound [I-].[I-].[I-].[I-].C12=CC=CC=C2C(C=CC=C2N(C3=CC=CC=C3O2)C)=CC=[N+]1CCC(=[N+](C)C)CCCC(=[N+](C)C)CC[N+](C1=CC=CC=C11)=CC=C1C=CC=C1N(C)C2=CC=CC=C2O1 JSBNEYNPYQFYNM-UHFFFAOYSA-J 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000003491 array Methods 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- SIPUZPBQZHNSDW-UHFFFAOYSA-N bis(2-methylpropyl)aluminum Chemical compound CC(C)C[Al]CC(C)C SIPUZPBQZHNSDW-UHFFFAOYSA-N 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 108091092328 cellular RNA Proteins 0.000 description 1
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 description 1
- 230000010428 chromatin condensation Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- UFULAYFCSOUIOV-UHFFFAOYSA-N cysteamine Chemical compound NCCS UFULAYFCSOUIOV-UHFFFAOYSA-N 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- WQABCVAJNWAXTE-UHFFFAOYSA-N dimercaprol Chemical compound OCC(S)CS WQABCVAJNWAXTE-UHFFFAOYSA-N 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- VHJLVAABSRFDPM-ZXZARUISSA-N dithioerythritol Chemical compound SC[C@H](O)[C@H](O)CS VHJLVAABSRFDPM-ZXZARUISSA-N 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 230000005670 electromagnetic radiation Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 108010070877 endodeoxyribonuclease uvrABC Proteins 0.000 description 1
- 230000006862 enzymatic digestion Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 108010032819 exoribonuclease II Proteins 0.000 description 1
- 108010079502 exoribonuclease T Proteins 0.000 description 1
- 201000003373 familial cold autoinflammatory syndrome 3 Diseases 0.000 description 1
- 108020002231 fibrillarin Proteins 0.000 description 1
- 102000005525 fibrillarin Human genes 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 239000005337 ground glass Substances 0.000 description 1
- 230000005283 ground state Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 210000003963 intermediate filament Anatomy 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 229910000358 iron sulfate Inorganic materials 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 210000005053 lamin Anatomy 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229960003151 mercaptamine Drugs 0.000 description 1
- 239000002082 metal nanoparticle Substances 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 210000000633 nuclear envelope Anatomy 0.000 description 1
- 238000001668 nucleic acid synthesis Methods 0.000 description 1
- 108020002020 oligoribonuclease Proteins 0.000 description 1
- 102000005549 oligoribonuclease Human genes 0.000 description 1
- 239000013307 optical fiber Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- ACVYVLVWPXVTIT-UHFFFAOYSA-M phosphinate Chemical compound [O-][PH2]=O ACVYVLVWPXVTIT-UHFFFAOYSA-M 0.000 description 1
- OJMIONKXNSYLSR-UHFFFAOYSA-N phosphorous acid Chemical compound OP(O)O OJMIONKXNSYLSR-UHFFFAOYSA-N 0.000 description 1
- 230000002186 photoactivation Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 238000011176 pooling Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108090000446 ribonuclease T(2) Proteins 0.000 description 1
- 108020005403 ribonuclease U2 Proteins 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 229910001961 silver nitrate Inorganic materials 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 235000010265 sodium sulphite Nutrition 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 108010050301 tRNA nucleotidyltransferase Proteins 0.000 description 1
- ISIJQEHRDSCQIU-UHFFFAOYSA-N tert-butyl 2,7-diazaspiro[4.5]decane-7-carboxylate Chemical compound C1N(C(=O)OC(C)(C)C)CCCC11CNCC1 ISIJQEHRDSCQIU-UHFFFAOYSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940071127 thioglycolate Drugs 0.000 description 1
- CWERGRDVMFNCDR-UHFFFAOYSA-M thioglycolate(1-) Chemical compound [O-]C(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-M 0.000 description 1
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 108060008226 thioredoxin Proteins 0.000 description 1
- 229940094937 thioredoxin Drugs 0.000 description 1
- HPGGPRDJHPYFRM-UHFFFAOYSA-J tin(iv) chloride Chemical compound Cl[Sn](Cl)(Cl)Cl HPGGPRDJHPYFRM-UHFFFAOYSA-J 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- TUQOTMZNTHZOKS-UHFFFAOYSA-N tributylphosphine Chemical compound CCCCP(CCCC)CCCC TUQOTMZNTHZOKS-UHFFFAOYSA-N 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6841—In situ hybridisation
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- G16B40/10—Signal processing, e.g. from mass spectrometry [MS] or from PCR
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B70/00—Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
- G16B15/10—Nucleic acid folding
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
- G16B15/30—Drug targeting using structural data; Docking or binding prediction
Definitions
- the present invention generally relates to genomics. Some embodiments are directed to imaging the 3D organization of the genome in the context of transcriptional activity and nuclear structures. In addition, certain embodiments are directed to chromatin organization and chromatin-nuclear-structure interactions as well as their relationship with transcription.
- Biochemical and imaging measurements have revealed complex chromatin structures across a wide range of scales.
- high-throughput chromosome conformation capture methods, such as Hi-C and other sequencing-based methods have greatly enriched knowledge of the 3D genome organization, revealing chromatin structures such as loops, domains, and compartments with a genome- wide view.
- Imaging-based approaches provide a direct measure of the spatial positions of chromatin loci in individual cells with a high detection efficiency.
- fluorescence in situ hybridization FISH
- CRISPR clustered regularly interspersed short palindromic repeats
- Chromatin imaging can also be combined with RNA and protein imaging to reveal the interplay between chromatin organization and transcriptional activity or interacting protein factors.
- current imaging methods are limited in throughput in the sequence space, traditionally allowing the study of only a few different genomic loci at a time. Genome-scale imaging would require a drastic increase in the number of genomic loci imaged in individual cells. Thus, new improvements are necessary.
- the present invention generally relates to genomics. Some embodiments are directed to imaging the 3D organization of the genome, or part of the genome, with high throughput in the sequence space. Some embodiments are directed to imaging the 3D organization of the genome, or part of the genome, in the context of transcriptional activity and nuclear structures. In addition, certain embodiments are directed to chromatin structures, 3D chromatin organizations, trans-chromosomal interactions and chromatin-nuclear- structure interactions as well as their relationship with transcription, etc.
- the subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.
- Certain aspects are generally directed to systems and methods of using multiplexed FISH, and in some cases using multiplexed error-robust FISH (MERFISH), to image chromatin, e.g., in a cell.
- MEFISH multiplexed error-robust FISH
- certain aspects are generally directed to systems and methods of imaging and/or determining at least 100 or at least 500 distinct genomic loci in a single cell.
- Some aspects are generally directed to systems and methods of using FISH to image chromatin, e.g., in a cell.
- the method comprises associating a plurality of nucleic acid targets of a genome with a plurality of codewords, wherein the codewords comprise a number of positions and values for each position; exposing a sample containing the genome to a plurality of nucleic acid probes; for each nucleic acid probe of the plurality of nucleic acid probes, determining binding of the nucleic acid probe within the sample; creating codewords corresponding to the binding of the plurality of nucleic acid probes within the sample; and determining the identities of the nucleic acid targets based on the codeword assigned.
- the method in another set of embodiments, comprises determining positions of nascent RNA within a nucleus; applying RNAse to the nucleus; and determining positions of DNA within the nucleus.
- the method comprises using MERFISH to image chromatin in a cell. In another set of embodiments, the method comprises imaging at least 100 or at least 500 distinct genomic loci in a single cell.
- the method comprises associating a plurality of nucleic acid targets of a genome with a plurality of codewords; exposing a sample containing a cell suspected of containing the genome to a plurality of nucleic acid probes, wherein at least some of the plurality of nucleic acid probes comprises a first portion comprising a target sequence and a second portion comprising one or more readout sequences, wherein each readout sequence represents a value of a position within the plurality of codewords; exposing the sample to a round of one or more adaptors, wherein each adaptor comprises a first portion substantially complementary to one of the readout sequences, and a second portion comprising one identification sequence; exposing the sample to a round of one or more readout probes to determine one or more identification sequences, wherein each readout probe comprises a first portion comprising a sequence substantially complementary to one of the identification sequences, and a second portion comprising a signaling entity; determining the signaling entity in at least some locations
- the method comprises associating a plurality of nucleic acid targets of a genome with a plurality of codewords; exposing a sample containing a cell suspected of containing the genome to a plurality of nucleic acid probes, wherein at least some of the plurality of nucleic acid probes comprises a first portion comprising a target sequence and a second portion comprising one or more readout sequences, wherein each readout sequence represents a value of a position within the plurality of codewords; exposing the sample to a round of one or more adaptors, wherein each adaptor comprises a first portion substantially complementary to one of the readout sequences, and a second portion comprising one identification sequence; exposing the sample to a round of one or more readout probes to determine one or more identification sequences, wherein each readout probe comprises a first portion comprising a sequence substantially complementary to one of the identification sequences, and a second portion comprising a signaling entity; determining the signaling entity in at least some locations
- the method comprises exposing a sample containing a cell suspected of containing the genome to a plurality of nucleic acid probes, wherein at least some of the plurality of nucleic acid probes comprises a first portion comprising a target sequence and a second portion comprising one or more readout sequences; exposing the sample to a round of one or more adaptors, wherein each adaptor comprises a first portion substantially complementary to one of the readout sequences, and a second portion comprising one identification sequence; exposing the sample to a round of one or more readout probes to determine one or more identification sequences, wherein each readout probe comprises a first portion comprising a sequence substantially complementary to one of the identification sequences, and a second portion comprising a signaling entity; determining the signaling entity in at least some locations in the sample; and inactivating the signaling entity in at least some locations in the sample; repeating the steps of exposing the sample to a round of one or more adaptors and one or more readout probes,
- the method in another set of embodiments, comprises exposing a sample containing a cell suspected of containing the genome to a plurality of nucleic acid probes, wherein at least some of the plurality of nucleic acid probes comprises a first portion comprising a target sequence and a second portion comprising one or more readout sequences; exposing the sample to a round of one or more readout probes to determine one or more readout sequences, wherein each readout probe comprises a first portion comprising a sequence substantially complementary to one of the readout sequences, and a second portion comprising a signaling entity; determining the signaling entity in at least some locations in the sample; and inactivating the signaling entity in at least some locations in the sample; repeating the steps of exposing the sample to a round of one or more readout probes, determining the signaling entity, and inactivating the signaling entity, wherein one or more distinct signaling entities are used in each of the rounds; determining nucleic acid targets in the sample based on the signaling entities determined in each
- the method comprises exposing a sample containing a cell suspected of containing the genome to a round of a plurality of nucleic acid probes, wherein at least some of the plurality of nucleic acid probes comprises a first portion comprising a target sequence and a second portion comprising a signaling entity; determining the signaling entity in at least some locations in the sample; and inactivating the signaling entity in at least some locations in the sample; repeating the steps of exposing the sample to a round of a plurality of nucleic acid probes, determining the signaling entity, and inactivating the signaling entity, wherein one or more distinct signaling entities are used in each of the rounds; determining nucleic acid targets in the sample based on the signaling entities determined in each round.
- the method includes associating a plurality of nucleic acid targets of a genome with a plurality of codewords, wherein the codewords comprise a number of positions and values for each position and the codewords form an error-checking and/or error-correcting code space, and wherein the plurality of nucleic acid targets are separated by at least 100,000 nucleotides within the genome; exposing a nucleus of a cell containing the genome to a plurality of nucleic acid probes, wherein at least some of the plurality of nucleic acid probes comprises a first portion comprising a target sequence and a second portion comprising one or more read sequences, wherein each read sequence represents a value of a position within the codewords; for each nucleic acid probe of the plurality of nucleic acid probes, determining binding of the nucleic acid probe within the nucleus; creating codewords corresponding to the binding of the plurality of nucleic acid probes within the nucleus, wherein the values
- the method comprises associating a plurality of nucleic acid targets of a genome with a plurality of codewords, wherein the codewords comprise a number of positions and values for each position and the codewords form an error-checking and/or error-correcting code space, and wherein the plurality of nucleic acid targets of the genome are distributed such that each chromosome of the genome contains no more than 200 nucleic acid targets; exposing a nucleus of a cell containing the genome to a plurality of nucleic acid probes, wherein at least some of the plurality of nucleic acid probes comprises a first portion comprising a target sequence and a second portion comprising one or more read sequences, wherein each read sequence represents a value of a position within the codewords; for each nucleic acid probe of the plurality of nucleic acid probes, determining binding of the nucleic acid probe within the nucleus; creating codewords corresponding to the binding of the plurality of nucleic acid probe
- the method comprises associating a plurality of between 500 and 1500 nucleic acid targets of a genome with a plurality of codewords, wherein the codewords comprise a number of positions and values for each position and the codewords form an error-checking and/or error-correcting code space; exposing a nucleus of a cell containing the genome to a plurality of nucleic acid probes, wherein at least some of the plurality of nucleic acid probes comprises a first portion comprising a target sequence and a second portion comprising one or more read sequences, wherein each read sequence represents a value of a position within the codewords; for each nucleic acid probe of the plurality of nucleic acid probes, determining binding of the nucleic acid probe within the nucleus; creating codewords corresponding to the binding of the plurality of nucleic acid probes within the nucleus, wherein the values of the digits of the codewords are based on the read sequences present on the nu
- the method includes associating a plurality of nucleic acid targets of a genome with a plurality of codewords, wherein the codewords comprise a number of positions and values for each position and the codewords form an error-checking and/or error-correcting code space, and wherein the plurality of nucleic acid targets are separated by at least 100,000 nucleotides within the genome; exposing a nucleus of a cell containing the genome to a plurality of nucleic acid probes; and determining a nucleic acid abundance and/or spatial distribution within the nucleus by determining binding of the plurality of nucleic acid probes within the nucleus using an error-checking and/or error-correcting detection technique.
- the method comprises associating a plurality of nucleic acid targets of a genome with a plurality of codewords; exposing a sample containing a cell suspected of containing the genome to a plurality of nucleic acid probes, wherein at least some of the plurality of nucleic acid probes comprises a first portion comprising a target sequence and a second portion comprising one or more read sequences, wherein each read sequence represents a value of a position within the plurality of codewords; exposing the sample to a plurality of adaptors, wherein at least some of the adaptors comprise a first portion substantially complementary to one or more of the read sequences, and a second portion comprising one or more identification sequences; exposing the sample to a round of one or more readout probes to determine one or more identification sequences, wherein at least some of the readout probes comprise a first portion comprising a sequence substantially complementary to one of the identification sequences, and a second portion comprising a signaling entity;
- the method comprises associating a plurality of nucleic acid targets of a genome with a plurality of codewords; exposing a sample containing a cell suspected of containing the genome to a plurality of nucleic acid probes, wherein at least some of the plurality of nucleic acid probes comprises a first portion comprising a target sequence and a second portion comprising one or more read sequences, wherein each read sequence represents a value of a position within the plurality of codewords; exposing the sample to a plurality of adaptors, wherein at least some of the adaptors comprise a first portion substantially complementary to one or more of the read sequences, and a second portion comprising one or more identification sequences; exposing the sample to a round of one or more readout probes to determine one or more identification sequences, wherein at least some of the readout probes comprise a first portion comprising a sequence substantially complementary to one of the identification sequences, and a second portion comprising a signaling entity;
- the method comprises determining positions of nascent RNA within a nucleus; determining positions of DNA within the nucleus; and determining positions of nuclear speckles within the nucleus.
- the method comprises determining positions of nascent RNA within a nucleus; determining positions of DNA within the nucleus; and determining positions of a protein within the nucleus. In still another set of embodiments, the method comprises determining positions of nascent RNA within a nucleus; determining positions of DNA within the nucleus; and determining positions of a nucleic acid within the nucleus, wherein the nucleic acid is not the nascent RNA or the DNA.
- Some aspects encompass methods of making one or more of the embodiments described herein. Also, some aspects encompasses methods of using one or more of the embodiments described herein.
- Figs. 1A-1I show genome-scale chromatin imaging, in accordance with certain embodiments
- Figs. 2A-2E show trans-chromosomal contacts enrichment, in another embodiment
- Figs. 3A-3H show genome-scale imaging of chromatin and transcription activity in the context of nuclear structures, in still another embodiment
- Figs. 4A-4F show trans-chromosomal interactions between active chromatin, in another embodiment
- Figs. 5A-5E illustrates a saturatable amplification system, in one embodiment
- Figs. 6A-6B show contact frequency matrices, in yet another embodiment
- Figs. 7A-7C show sub-chromosomal structures derived from genome-scale imaging and comparison with ensemble Hi-C data, in still another embodiment
- Fig. 8 shows reproducibility of the chromatin imaging experiments between replicates, in another embodiment
- Figs. 9A-9B show distinct spatial distributions in single cells, in certain embodiments.
- Figs. 10A-10B show nascent RNA transcript imaging, in yet other embodiments
- Fig. 11 shows the association of compartment-B loci with nuclear lamina, in certain embodiments
- Fig. 12 shows the association of compartment-A loci with nuclear speckles, in some embodiments
- Figs. 13A-13C show changes in nuclear lamina and nuclear speckle association upon transcription inhibition
- Fig. 14 shows the local density of trans-chromosomal A loci near each imaged locus, in yet another embodiment
- Figs. 15A-15B show enrichment of active-active trans-chromosomal interactions among chromatin loci, in still another embodiment;
- Figs. 16A-16B show the enrichment of active-active trans-chromosomal interactions, in yet another embodiment;
- Figs. 17A-17M show high-resolution whole-chromosome tracing by sequential hybridization and characterization of chromatin domains in single cells, in one embodiment
- Figs. 18A-18I show the compartment structure in single chromosomes and relationship between transcription activity and local chromatin content, in another embodiment
- Figs. 19A-19H show the dependence of domain-domain interaction on their A/B composition and genomic distance, in yet another embodiment
- Figs. 20A-20H show genome-scale chromatin imaging by massively multiplexed, combinatorial FISH, in still another embodiment
- Figs. 21A-21E show enrichment of active-active chromatin interactions in trans- chromosomal interactions, according to one embodiment
- Figs. 22A-22J show multi-modal genome-scale imaging of chromatin and transcription activity in the context of nuclear structures, in accordance with another embodiment
- Figs. 23A-23D show the correlation between transcriptional activity and local enrichment of /ran.s-chromosomal active chromatin, in yet another embodiment
- Figs. 24A-24N show high-resolution whole-chromosome tracing by sequential hybridization, and ensemble statistics of Chr21 structural features in comparison with Hi-C, in still another embodiment
- Figs. 25A-25G show ensemble A/B compartment analyses for Chr21 and Chr2, in yet another embodiment
- Figs. 26A-26J show measurements for RNA and DNA FISH probe crosstalk, in still another embodiment
- Figs. 27A-27J show genome-scale imaging by combinatorial FISH: localization error, reproducibility, and comparison with Hi-C, in one embodiment
- Figs. 28A-28B show that compartment-A and compartment-B loci display distinct spatial distributions in the nucleus, according to another embodiment
- Figs. 29A-29F show the effect of transcriptional inhibition on the trans-chromosome chromatin interactions and the nuclear body association rates of chromatin loci, in yet another embodiment.
- Figs. 30A-30D show enrichment of /rani-chromosomal active chromatin interactions in different nuclear environments, in still another embodiment.
- the present invention generally relates to genomics. Some embodiments are directed to imaging the 3D organization of the genome, or part of the genome, with high throughput in the sequence space. Some embodiments are directed to imaging the 3D organization of the genome, or part of the genome, in the context of transcriptional activity and nuclear structures. In addition, certain embodiments are directed to chromatin structures, 3D chromatin organizations, trans-chromosomal interactions and chromatin-nuclear- structure interactions as well as their relationship with transcription, etc. In addition, various embodiments are directed to imaging methods that allow mapping of the 3D organization of the genome, or part of the genome, in the context of nuclear structures and transcriptional activity.
- Some embodiments are directed to massively multiplexed fluorescence in situ hybridization methods for imaging chromatin loci and/or nascent RNA transcripts at the chromosome or genome scale.
- simultaneous imaging of hundreds of genomic loci can be performed.
- simultaneous imaging of -1000 genomic loci and/or transcriptional activities of -1000 genes within these loci together with various nuclear structures can be performed.
- chromatin domains and compartments can be observed.
- extensive trans-chromosomal interactions that were enriched for active chromatin interactions in a transcription-correlated manner can be observed.
- transcription-dependent chromatin interactions with nuclear speckles and nuclear lamina across the genome can be observed.
- 3D three-dimensional (3D) organization of chromatin regulates many genome functions.
- An understanding of 3D genome organization is hindered by the lack of tools that allow direct visualization of chromatin organization at the chromosome scale and genome scale in its native context.
- a multiplexed FISH approach by sequential imaging over multiple hybridization rounds, for example, such that each round targets one or two or three genomic loci using one- or two- or three-color imaging.
- Described in other embodiments are a combinatorial FISH approach in many chromatin loci are imaged simultaneously in each round and their distinct identities are determined based on the combinations of rounds they appear in. This is generally based on MERFISH and other approaches, e.g., as discussed in Int. Pat. Apl. Pub.
- Some aspects are generally directed to systems and methods of using multiplexed FISH, or other techniques, in some cases using MERFISH, including those described herein, to image chromosomes or chromatin, e.g., in a cell.
- certain embodiments are generally directed to systems and methods of imaging and/or determining at least 100 distinct genomic loci, at least 500 distinct genomic loci, or at least 1,000 distinct genomic loci, etc. in a single cell.
- other parts of the cell, or the nucleus may be determined, for example, RNA present within the nucleus, e.g., nascent RNA, nuclear speckles, nucleoli, nuclear lamina, other nuclear structures or proteins, etc.
- the positions of chromosomes or chromatin, the nascent RNAs, nuclear speckles, nucleoli, and/or nuclear lamina may be determined.
- Certain embodiments are directed to determining a sample, which may include a cell culture, a suspension of cells, a biological tissue, a biopsy, an organism, or the like.
- the sample can also be cell-free but nevertheless contain nucleic acids in some cases.
- the cell may be a human cell, or any other suitable cell, e.g., a mammalian cell, a fish cell, an insect cell, a plant cell, or the like. More than one cell may be present in some cases.
- the targets to be determined can include nucleic acids, proteins, or the like. For example, these may be present within the nucleus of cells within the sample.
- chromatin within a cell can be determined, for instance, relative to nuclear structures of the cell, including nuclear speckles, nucleoli, nuclear lamina, or nuclear structures or proteins.
- chromatin loci and/or RNA transcripts may be determined within the cell, e.g., at the chromosome or genome scale.
- the nucleic acids within a cell are to be determined. These typically include DNA (e.g., genomic DNA, which may be present in the form of chromatin, e.g., packaged together with proteins such as histones) and RNA (e.g., at the start of the transcription phase when the DNA is transcribed into RNA; this RNA within the nucleus is sometimes referred to as nascent RNA).
- DNA e.g., genomic DNA, which may be present in the form of chromatin, e.g., packaged together with proteins such as histones
- RNA e.g., at the start of the transcription phase when the DNA is transcribed into RNA; this RNA within the nucleus is sometimes referred to as nascent RNA.
- the DNA is highly packed within the nucleus of the cell, making it substantially more difficult to determine its structure.
- the DNA may be packed within the cell as chromosomes or chromatin, and such DNA may often be entangled or packed closely together within the nucleus.
- the DNA targets may be selected to be spatially separated.
- the sample is subjected to multiple rounds of hybridization with nucleic acid probes, where one or more rounds round targets one or more target nucleic acids with single-color or multi-color imaging.
- the identities of the target nucleic acids are determined based on which round and/or which color channel they are imaged.
- the positions of the target nucleic acids are determined. In some cases, at least 50, at least 100, at least 500, at least 1000, at least 5000, or at least 10,000 target nucleic acids are determined.
- the target nucleic acids are genomic loci. In some cases, the target nucleic acids are genomic loci and/or nascent RNA transcripts. In some cases, the positions of the genomic loci are used to determine the three-dimensional organization of chromatin or the three-dimensional organization of the genome in the cell.
- primary nucleic acid probes able to target nucleic acids within a cell are designed.
- the probes each contain a target sequence that binds to one of the target nucleic acids.
- the probes may also contain a portion that comprises one or more “readout sequences” that can be used to determine the identity and the position of the primary nucleic acid probes.
- the primary nucleic acid probes may contain a plurality of readout sequences. These can be individually read using one or multiple rounds of secondary nucleic acid probes, called readout probes, that can bind to a readout sequence of the primary nucleic acid probe.
- the readout probes may also contain a signaling entity, such as a florescent entity, e.g., that can be determined using various microscopy techniques.
- a signaling entity such as a florescent entity, e.g., that can be determined using various microscopy techniques.
- the multiple rounds of readout probes may be applied sequentially, such that one type of readout probe is applied to a sample and fluorescence within the sample determined, then the readout probe, or the signaling entity on the readout probe, is inactivated or removed and the next type of readout probe applied.
- locations within the sample may be associated with a plurality of readout probes, and this information may be digitized for analysis.
- the multiple rounds of readout probes may be applied sequentially, such that more than one type of readout probes is applied to a sample in each round and/or fluorescence within the sample is determined using multi-color imaging, then the readout probes, and/or the signaling entities on the readout probes, are inactivated or removed and the next set of more than one types of readout probes applied.
- locations within the sample may be associated with a plurality of readout probes, and this information may be digitized for analysis.
- the locations of the primary nucleic acid probes, and the target nucleic acids may be determined using one or multiple rounds of readout probes. For example, there may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, etc. rounds of readout probes.
- a sample may be exposed to multiple rounds of applying readout probes, determining the probes within the sample (e.g., using a signaling entity, such as is described herein), and removing or inactivating the secondary nucleic acid probes.
- more than one round of identical readout probes may be used, for example, to determine whether any degradation and/or movement has occurred in the sample, e.g., over time, due to the effects of supplying multiple rounds of nucleic acids or other chemicals, as a control, etc.
- the sample is subjected to multiple rounds of hybridization with nucleic acid probes and each round is subject to single-color or multi-color imaging.
- the identities of the target nucleic acids are determined based on which combination of rounds and/or color channel they are imaged.
- the positions of the target nucleic acids are determined. In some cases, at least 50, at least 100, at least 500, at least 1000, at least 5000, or at least 10,000 target nucleic acids are determined.
- the target nucleic acids are genomic loci. In some cases, the target nucleic acids are genomic loci and/or nascent RNA transcripts. In some cases, the positions of the genomic loci are used to determine the three-dimensional organization of chromatin or the three-dimensional organization of the genome in the cell.
- primary nucleic acid probes also called encoding probes
- the probes each comprise a target sequence that binds to one of the target nucleic acids.
- the probes may also contain a portion that comprises one or more “readout sequences” that can be used to determine the identity and the position of the primary or encoding nucleic acid probes.
- the primary or encoding nucleic acid probes may contain a plurality of readout sequences. These can be individually read using one or multiple rounds of readout probes, that can bind to a readout sequence of the primary or encoding nucleic acid probe.
- the readout probes may also contain a signaling entity, such as a florescent entity, e.g., that can be determined using various microscopy techniques.
- a signaling entity such as a florescent entity, e.g., that can be determined using various microscopy techniques.
- the multiple rounds of readout probes may be applied sequentially, such that one type of readout probe is applied to a sample and fluorescence within the sample determined, then the readout probe, or the signaling entity on the readout probe, is inactivated or removed and the next type of readout probe applied.
- locations within the sample may be associated with a plurality of readout probes, and this information may be digitized for analysis.
- the multiple rounds of readout probes may be applied sequentially, such that more than one type of readout probes are applied to a sample in each round and fluorescence within the sample determined using multi-color imaging, then the readout probes, or the signaling entities on the readout probes, are inactivated or removed and the next set of more than one types of readout probes applied.
- locations within the sample may be associated with a plurality of readout probes, and this information may be digitized for analysis.
- the locations of the primary or encoding nucleic acid probes, and the target nucleic acids may be determined using one or multiple rounds of readout probes. For example, there may be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 16, at least 20, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, etc. rounds of readout probes.
- a sample may be exposed to multiple rounds of applying readout probes, determining the probes within the sample (e.g., using a signaling entity, such as is described herein), and removing or inactivating the secondary nucleic acid probes.
- the primary or encoding nucleic acid probes may be designed in some embodiments such that different targets within the sample are determinable using different combination of readout sequences, without necessarily requiring each of the readout sequences to be unique. As a non-limiting example, with 4 possible readout sequences A, B, C, and D, up to 6 different targets may be identified if the set of primary or encoding nucleic acid probes targeting each nucleic acid target only contains 2 readout sequences, e.g., corresponding to AB, AD, CB, CD, AC, and DB.
- not all of the possible combinations of readout sequences will be used. Instead, some of the combinations may not be assigned to any target in the nucleus, e.g., no primary or encoding nucleic acid probes having those combinations may be used.
- the valid combinations of readout sequences used in the primary or encoding nucleic acid probes may be arranged so as to form an error-checking and/or an error correcting code space. Using such a method, determinations of readout sequences within the sample that do not correspond with a valid primary nucleic acid probe may be determined using error-checking to be in error, and in some cases, can even be corrected using error correction, e.g., to correspond to a valid primary nucleic acid probe.
- RNA e.g., nascent RNA
- the targets of the primary or encoding nucleic acid probes can be chosen such that binding within the nucleus occurs in a spatially separated manner.
- the targets can be chosen such that they are separated in genomic space, e.g., separated by at least 10,000 bp at least 30,000 bp at least 100,000 bp, at least 300,000 bp, at least 1,000,000 bp within the genome, or such that the genomic space contains no more than 100, no more than 200, no more than 300, no more than 500, no more than 1000, no more than 5000, no more than 10,000, no more than 50,000, no more than 100,000 nucleic acid targets.
- more than one type of fluorescent probe or “color” can also be used, e.g., to allow more targets to be determined within the nucleus.
- the cell and/or nucleus may also be modified to allow such probes to reach the nucleic acids therein.
- the cells may be permeabilized or “fixed” to allow entry of nucleic acid probes.
- the DNA may be denatured in some embodiments, e.g., by applying heat, in order to allow more ready access to the DNA by the primary or encoding nucleic acid probes. This is not typically performed for RNA determinations, as RNA is single- stranded while DNA is usually double- stranded.
- the RNA within the nucleus must be removed and/or inactivated, for example, to prevent probes targeting DNA from binding to the RNA.
- an enzyme such as an RNase may be applied to the nucleus to prevent RNA from interfering with DNA determination.
- the RNA within the nucleus may also be determined. This may be particularly valuable, e.g., when studying the spatial locations of DNA and RNA within a nucleus, and how they relate to each other.
- the RNA within a nucleus may be determined, e.g., analogously to that described above for genomic DNA, prior to removal or inactivation of the RNA as described above.
- proteins within a cell may also be determined. Examples include, but are not limited to nuclear speckle, nucleolis, or histone proteins.
- a variety of methods for determining proteins can be used. For instance, in one set of embodiments, immunofluorescence assay can be used. In another set of embodiments, a “sandwich assay” may be used, where a primary antibody able to specifically bind to a nuclear protein is applied, then a secondary antibody able to specifically bind to the primary antibody is used, where the secondary antibody contains a signaling entity, such as a florescent entity.
- proteins and nucleic acids within the nucleus of a cell may be determined, e.g., spatially.
- nucleic acids such as genomic DNA and/or nascent RNA
- various aspects are directed to various systems and methods for nucleic acids.
- one, two, or more of DNA, RNA, and protein within a cell may be determined.
- the nucleic acids within a nucleus to be determined may include, for example, DNA (for example, genomic DNA), RNA, or other nucleic acids that are present within a cell (or other sample).
- the nucleic acids may be endogenous to the cell, or added to the cell.
- the nucleic acid may be viral, or artificially created.
- the nucleic acid to be determined may be expressed by the cell.
- the nucleic acid is RNA in some embodiments.
- the RNA may be coding and/or non-coding RNA.
- the RNA may encode a protein.
- Non-limiting examples of RNA that may be studied within the cell include mRNA, siRNA, rRNA, miRNA, tRNA, IncRNA, snoRNAs, snRNAs, exRNAs, piRNAs, or the like.
- all, or at least a significant portion of the genome of a cell may be determined.
- the determined genomic segments may be continuous or interspersed on the genome.
- at least 4 genomic segments are determined within a cell, and in some cases, at least 3, at least 4, at least 7, at least 8, at least 12, at least 14, at least 15, at least 16, at least 22, at least 30, at least 31, at least 32, at least 50, at least 63, at least 64, at least 72, at least 75, at least 100, at least 127, at least 128, at least 140, at least
- At least 256, at least 500, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 4,000, at least 5,000, at least 7,500, at least 10,000, at least 12,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 40,000, at least 50,000, at least 75,000, or at least 100,000 genomic segments may be determined within a cell.
- the entire genome of a cell may be determined. It should be understood that the genome generally encompasses all DNA molecules produced within a cell, not just chromosome DNA. Thus, for instance, the genome may also include, in some cases, mitochondria DNA, chloroplast DNA, plasmid DNA, etc., e.g., in addition to (or instead of) chromosome DNA.
- At least about 0.01%, at least about 0.1%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or 100% of the genome of a cell may be determined.
- RNA within the nucleus e.g., nascent RNA
- enough of the RNA present within a cell may be determined so as to produce a partial or complete transcriptome of the cell.
- RNAs e.g., mRNAs, nascent RNA, etc.
- RNAs are determined within a cell, or within the nucleus of the cell, and in some cases, at least 3, at least 4, at least 7, at least 8, at least 12, at least 14, at least 15, at least 16, at least 20, at least 22, at least 30, at least 31, at least 32, at least 50, at least 63, at least 64, at least 72, at least 75, at least 100, at least 127, at least 128, at least 140, at least 255, at least
- RNAs may be determined within a cell, or within the nucleus of the cell.
- the transcriptome of a cell may be determined. It should be understood that the transcriptome generally encompasses all RNA molecules produced within a cell, not just mRNA. Thus, for instance, the transcriptome may also include rRNA, tRNA, siRNA, etc. in certain instances. In some embodiments, at least about 0.01%, at least about 0.1%, at least about 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the transcriptome of a cell may be determined. In addition, in some cases, the transcriptome of the nucleus of a cell may be determined.
- targets to be determined can include targets that are linked to nucleic acids, proteins, or the like.
- a binding entity able to recognize a target may be conjugated to a nucleic acid probe.
- the binding entity may be any entity that can recognize a target, e.g., specifically or non- specifically.
- Non-limiting examples include enzymes, antibodies, receptors, complementary nucleic acid strands, aptamers, or the like.
- an oligonucleotide-linked antibody may be used to determine a target. The target may bind to the oligonucleotide-linked antibody, and the oligonucleotides determined as discussed herein.
- the determination of targets, such as nucleic acids within the cell or other sample may be qualitative and/or quantitative.
- the determination may also be spatial, e.g., the position of the nucleic acids, or other targets, within the cell or other sample may be determined in two or three dimensions.
- the positions, number, and/or concentrations of nucleic acids, or other targets, within the cell or other sample may be determined.
- the DNA within the nucleus of a cell e.g., the genomic DNA, of the cell may be studied, for example, using nucleic acid probes such as discussed herein, e.g., including using sequential imaging or using combinatorial imaging with error-detecting and/or an error-correcting codes.
- the DNA targets or the codes associated with the DNA targets within a cell, or within the nucleus of a cell may be chosen such that the targets are spatially separated in each round of imaging, e.g., in genomic space, or in physical space based on knowledge of chromatin organization, for example, such as the organization of chromosomes into compact territories. This may be useful, for example, to be able to identify different targets within the cell of the nucleus of the cell, e.g., using techniques such as those discussed herein.
- the targets within the genomic space may be selected using any suitable technique, e.g., randomly, or having a substantially uniform probabilistic distribution, etc. In certain embodiments, the targets may be selected individually to ensure spatial separation. In addition, in some embodiments, the targets may be selected to be those targets of interest within the genome, e.g., for a particular study. For instance, in some embodiments, the targets may be chosen within a genomic space such that a nucleus will have no more than a certain number of nucleic acid targets.
- the targets may be chosen such that the genomic space contains no more than 100,000, no more than 10,000, no more than 8,000, no more than 6,000, no more than 5,000, no more than 4,000, no more than 3,000, no more than 2,000, no more than 1,500, no more than 1,000, no more than 900, no more than 800, no more than 700, no more than 600, no more than 500, no more than 400, no more than 300, no more than 200, no more than 100 nucleic acid targets, no more than 30 nucleic acid targets, or no more than 10 nucleic acid targets.
- the targets may be chosen such that the genomic space contains at least 10, at least 30, at least 50, at least 100, at least 200, at least 300, at least 500, at least 1,000, at least 1,500, at least 2,000, at least 3,000, at least 5,000, at least 10,000, at least 100,000, etc. nucleic acid targets. Combinations of any of these are also possible in certain embodiments, e.g., there may be between 30 and 100, between 3,000 and 5,000, between 500 and 1,500, etc. nucleic acid targets. Such targets may be chosen, e.g., selectively, randomly, etc., as is discussed herein.
- the targets may be selected such that the chromosomes within the genome have no more than a certain number of nucleic acid targets (e.g., genomic loci).
- the targets may be chosen such that each chromosome has no more than 10,000, no more than 1000, no more than 500, no more than 400, no more than 300, no more than 200, no more than 150, no more than 125, no more than 100, no more than 90, no more than 80, no more than 70, no more than 60, no more than 50, no more than 40, no more than 30, no more than 20, or no more than 10 nucleic acid targets.
- the targets may be chosen to have at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 200, at least 300, at least 400, at least 1,000, at least 10,000 etc. nucleic acid targets.
- combinations of these may be selected, e.g., a chromosome may have between 30 and 50, between 40 and 100, between 50 and 60, between 30 and 80, etc., nucleic acid targets.
- different chromosomes may independently have the same or different numbers of nucleic acid targets, e.g., including the ranges described herein.
- nucleic acid targets within the genome may be selected to have specific structural or functional properties, such as promoters, enhancers, and loci bound by specific nuclear architecture proteins.
- nucleic acid targets may be nucleic acid targets that are unique to their respective chromosomes.
- the targets may be selected to be separated by a minimum of a certain number of nucleotides, e.g., to facilitate a distribution of targets that are spatially separated.
- targets may be selected within the genome such that every target is separated by at least 1,000, at least 3,000, at least 5,000, at least 10,000, at least 30,000, at least 50,000, at least 100,000, at least 300,000, at least 500,000, at least 1,000,000, at least 3,000,000, at least 5,000,000, at least 10,000,000, etc. nucleotides.
- the targets may be selected within the genome such that every target is separated by no more than 10,000,000, no more than 5,000,000, no more than 3,000,000, no more than 1,000,000, no more than 500,000, no more than 300,000, no more than 100,000, no more than 50,000, no more than 30,000, no more than 10,000 nucleotides. Combinations of any of these are also possible in certain embodiments, e.g., the targets may be separated by between 30,000 and 100,000 between 3,000,000 and 5,000,000, between 500,000 and 1,000,000 etc. nucleotides. Such targets may be chosen, e.g., selectively, randomly, etc., as is discussed herein.
- the RNA within the nucleus of a cell e.g., the nascent RNA, of the cell may be studied, for example, instead of or in addition to the DNA within the nucleus as described above.
- the RNA within a nucleus may be determined, then the DNA within the nucleus may be determined.
- the RNA may be removed or inactivated before determining the DNA. This may facilitate separation of the DNA and RNA determinations, e.g., since no RNA signal will be present that could complicate the DNA determination.
- methods of removing or inactivating RNA include the use of RNases such as endoribonucleases or exoribonucleases.
- RNase A RNase A, RNase H, RNase III, RNase L, RNase P, RNase PhyM, RNase Tl, RNase T2, RNase U2, RNase V, PNPase, RNase PH, RNase R, RNase D, RNase T, oligoribonuclease, exoribonuclease I, exoribonuclease II, or the like.
- the DNA may be determined before the RNA, and/or both may be determined simultaneously.
- DNA may be removed or inactivated after determination using DNases such as an exodeoxyribonuclease or an endodeoxyribonuclease. Examples include, but are not limited to, deoxyribonuclease I (DNase I), deoxyribonuclease II (DNase II), DNase IV, UvrABC endonuclease, or the like.
- DNase I deoxyribonuclease I
- DNase II deoxyribonuclease II
- DNase IV DNase IV
- UvrABC endonuclease or the like.
- the DNA may be degraded via exposure to a restriction endonuclease. Many such nucleases are available commercially.
- the RNA within the nucleus may be determined using any suitable technique, and may be determined using the same or different techniques than used to determine the DNA within the nucleus.
- the RNA can be determined using MERFISH. See, e.g., Int. Pat. Apl. Pub. No. WO 2016/018960, entitled “Systems and Methods for Determining Nucleic Acids”; and Int. Pat. Apl. Pub. No. WO 2016/018963, entitled “Probe Library Construction,” each incorporated herein by reference in its entirety.
- the RNA may be determined using a plurality of nucleic acid probes, e.g., as discussed herein.
- RNA may be determined using nucleic acids such as encoding nucleic acid probes, primary amplifier nucleic acids, secondary amplifier nucleic acids, etc., as described below.
- the nucleic acid probes may define an error-detecting and/or an error-correcting code, e.g., as discussed herein.
- DNA such as genomic DNA may be determined using nucleic acids such as encoding nucleic acid probes, primary amplifier nucleic acids, secondary amplifier nucleic acids, etc., as described herein.
- the nucleic acid probes may define an error-detecting and/or an error-correcting code, e.g., as discussed herein.
- proteins within the nucleus of a cell may be studied, e.g., in addition to nucleic acids present within the nucleus, using techniques such as those described above.
- proteins that can be studied include, but are not limited to nuclear speckles, nucleoli, the nuclear lamina, or histone proteins, etc.
- Speckles are structures that are enriched in pre-messenger RNA splicing factors and may be located in the interchromatin regions of the nucleoplasm of mammalian cells.
- Nucleoli are structures formed around the highly transcribed genomic loci encoding ribosomal RNA (rRNA), and may be enriched for rRNA and the transcriptional machinery associated with it.
- the nuclear lamina is a protein structure associated with the inner nuclear membrane, and may be enriched for intermediate filaments (lamins), as well as chromatin that is transcriptionally inactive.
- Histones are proteins used to wrap or fold the DNA into more compact complexes within the nucleus, forming chromatin.
- an immunofluorescence assay may be used.
- a “sandwich assay” may be used, where a primary antibody able to specifically bind to a nuclear protein is applied, then a secondary antibody able to specifically bind to the primary antibody is used, where the secondary antibody contains a signaling entity, such as a florescent entity or an oligonucleotide that can be detected, e.g., using a complementary oligonucleotide linked to a fluorescent entity.
- a signaling entity such as a florescent entity or an oligonucleotide that can be detected, e.g., using a complementary oligonucleotide linked to a fluorescent entity.
- proteins and nucleic acids within the nucleus of a cell may be determined, e.g., spatially.
- nucleic acid probes may be used to determine one or more targets within a cell or other sample, e.g., within the nucleus of the cell.
- the probes may comprise nucleic acids (or entities that can hybridize to a nucleic acid, e.g., specifically) such as DNA, RNA, LNA (locked nucleic acids), PNA (peptide nucleic acids), and/or combinations thereof.
- nucleic acid probes include, but are not limited to, those described in Int. Pat. Apl. Pub.
- WO 2016/018960 entitled “Systems and Methods for Determining Nucleic Acids”; and Int. Pat. Apl. Pub. No. WO 2016/018963, entitled “Probe Library Construction,” each incorporated herein by reference in its entirety.
- additional components may also be present within the nucleic acid probes, e.g., as discussed below.
- any suitable method may be used to introduce nucleic acid probes into a cell, e.g., to target its nucleus.
- the cell is fixed prior to introducing the nucleic acid probes, e.g., to preserve the positions of the nucleic acids or other targets within the cell, e.g., within its nucleus.
- Techniques for fixing cells are known to those of ordinary skill in the art.
- a cell may be fixed using chemicals such as formaldehyde, paraformaldehyde, glutaraldehyde, ethanol, methanol, acetone, acetic acid, or the like.
- a cell may be fixed using HEPES -glutamic acid buffer-mediated organic solvent (HOPE).
- HOPE HEPES -glutamic acid buffer-mediated organic solvent
- the cell (or other sample) may be fixed more than once, e.g., during relatively long experiments.
- the sample may be re-fixed after the start of an experiment, e.g., after exposing the nucleus of the cell to the plurality of nucleic acid probes.
- the cell or other sample may be fixed at least once every 7 days, at least once every 4 days, at least once every 2 days, at least once every day, at least once every 12 hours, at least once every 6 hours, at least once every 3 hours, etc. In some cases, this may be done between various rounds, e.g., of exposure to nucleic acid probes (e.g., primary or secondary nucleic acid probes), etc.
- the sample may be fixed a certain number of times, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or any other suitable number of times. If multiple fixes occur, these may independently use the same or different fixation techniques.
- the nucleic acid probes may be introduced into the cell (or other sample) using any suitable method.
- the cell may be sufficiently permeabilized such that the nucleic acid probes may be introduced into the cell by flowing a fluid containing the nucleic acid probes around the cells.
- the cells may be sufficiently permeabilized as part of a fixation process; in other embodiments, cells may be permeabilized by exposure to certain chemicals such as ethanol, methanol, Triton, or the like.
- techniques such as electroporation or microinjection may be used to introduce nucleic acid probes into a cell or other sample.
- nucleic acid probes that are introduced into a cell (or other sample).
- the probes may comprise any of a variety of entities that can hybridize to a nucleic acid, typically by Watson-Crick base pairing, such as DNA, RNA, LNA, PNA, etc., depending on the application.
- the nucleic acid probe typically contains a target sequence that is able to bind to at least a portion of a target, e.g., a target nucleic acid.
- the binding may be specific binding (e.g., via complementary binding).
- the target sequence may be able to bind to a specific target (e.g., nascent RNA, genomic DNA, an mRNA, or other nucleic acids as discussed herein).
- the nucleic acid probe may also contain one or more readout sequences, as discussed below.
- more than one type of nucleic acid probe may be applied to a sample, e.g., sequentially or simultaneously. For example, there may be at least 2, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 300, at least 1,000, at least 3,000, at least 10,000, at least 30,000, at least 100,000, at least 300,000, at least 1,000,000 distinguishable nucleic acid probes that are applied to a sample, e.g., to a cell to target its nucleus. In some cases, the nucleic acid probes may be added sequentially. However, in some cases, more than one nucleic acid probe may be added simultaneously.
- the nucleic acid probe may include one or more target sequences, which may be positioned anywhere within the nucleic acid probe.
- the target sequence may contain a region that is substantially complementary to a portion of a target, e.g., a target nucleic acid, which may be within the nucleus.
- the portions may be at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary, e.g., to produce specific binding.
- complementarity is determined on the basis of Watson-Crick nucleotide base pairing.
- the target sequence may be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 65, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 nucleotides in length.
- the target sequence may be no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, no more than 175, no more than 150, no more than 125, no more than 100, be no more than 75, no more than 60, no more than 65, no more than 60, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 20, or no more than 10 nucleotides in length.
- the target sequence may have a length of between 10 and 30 nucleotides, between 20 and 40 nucleotides, between 5 and 50 nucleotides, between 10 and 200 nucleotides, or between 25 and 35 nucleotides, between 10 and 300 nucleotides, etc.
- the nucleic acid targets or the codes associated with the nucleic acid targets within a cell or the nucleus of a cell may be chosen such that the targets are spatially separated in each round of imaging, e.g., in genomic space , or in physical space based on previous knowledge of chromatin organization such as the organization of chromosomes into compact territories.
- the target sequence of a nucleic acid probe may be determined with reference to a target suspected of being present within a cell or other sample, e.g., within the nucleus of the cell.
- a target nucleic acid to a protein e.g., nuclear speckles, nuclear lamina, etc.
- the protein’s sequence e.g., by determining the nucleic acids that are expressed to form the protein.
- only a portion of the nucleic acids encoding the protein are used, e.g., having the lengths as discussed above.
- More than one target sequence that can be used to identify a particular target may be used, in accordance with certain embodiments.
- multiple probes can be used, sequentially and/or simultaneously, that can bind to or hybridize to the same or different regions of the same target.
- Hybridization typically refers to an annealing process by which complementary single- stranded nucleic acids associate through Watson-Crick nucleotide base pairing (e.g., hydrogen bonding, guanine-cytosine and adenine-thymine) to form double- stranded nucleic acid.
- a nucleic acid probe may also comprise one or more “readout” sequences.
- the readout sequences may be used, to identify the nucleic acid probe, e.g., through association with signaling entities, as discussed below.
- the nucleic acid probe may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more, 20 or more, 24 or more, 32 or more, 40 or more, 48 or more, 50 or more, 64 or more, 75 or more, 100 or more, 128 or more readout sequences.
- the readout sequences may be positioned anywhere within the nucleic acid probe. If more than one readout sequence is present, the readout sequences may be positioned next to each other, and/or interspersed with other sequences.
- the readout sequences may be of any length. If more than one readout sequence is used, the readout sequences may independently have the same or different lengths. For instance, the readout sequence may be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 65, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 nucleotides in length.
- the readout sequence may be no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, no more than 175, no more than 150, no more than 125, no more than 100, be no more than 75, no more than 60, no more than 65, no more than 60, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 20, or no more than 10 nucleotides in length.
- the readout sequence may have a length of between 10 and 30 nucleotides, between 20 and 40 nucleotides, between 5 and 50 nucleotides, between 10 and 200 nucleotides, or between 25 and 35 nucleotides, between 10 and 300 nucleotides, etc.
- the readout sequence may be arbitrary or random in some embodiments.
- the readout sequences are chosen so as to reduce or minimize homology with other components of the cell or other sample, e.g., such that the readout sequences do not themselves bind to or hybridize with other nucleic acids suspected of being within the cell or other sample.
- the homology may be less than 10%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.
- the readout sequences may exhibit no specific binding towards each other, and/or such that none of the readout sequences in the population of readout sequences has a complementary of more than 5, 6, 7, 8, 9, 10, etc. nucleotides to another readout sequence within the population of readout sequences.
- a population of nucleic acid probes may contain a certain number of readout sequences, which may be the same as the number of nucleic acid targets to be determined in the sample, for example, with each unique readout sequence corresponding to a unique target.
- a population of nucleic acid probes may contain a certain number of readout sequences, which may be less than the number of nucleic acid targets to be determined in the sample.
- a population of nucleic acid probes may target 12 different nucleic acid targets, yet contain no more than 8 readout sequences.
- a population of nucleic acid probes may target 140 different nucleic acid targets, yet contain no more than 16 readout sequences.
- Different nucleic acid targets may be separately identified by using different combinations of readout sequences within each probe.
- the population of nucleic acid probes may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, etc. or more readout sequences.
- a population of nucleic acid probes may each contain the same number of readout sequences, although in other cases, there may be different numbers of readout sequences present on the various probes.
- a first nucleic acid probe may contain a first target sequence, a first readout sequence, and a second readout sequence
- a second, different nucleic acid probe may contain a second target sequence, the same first readout sequence, but a third readout sequence instead of the second readout sequence.
- Such probes may thereby be distinguished by determining the various readout sequences present or associated with a given probe or location, as discussed herein.
- the probes can be sequentially identified and encoded using “codewords,” as discussed below.
- the codewords may also be subjected to error detection and/or correction.
- a first population of nucleic acid probes may contain a first target sequence, a first readout sequence, and a second readout sequence
- a second, different population of nucleic acid probes may contain a second target sequence, the same first readout sequence, but a third readout sequence instead of the second readout sequence.
- Such probes may thereby be distinguished by determining the various readout sequences present or associated with a given probe or location, as discussed herein.
- the populations of probes can be sequentially identified and encoded using “codewords,” as discussed below.
- the codewords may also be subjected to error detection and/or correction.
- the population of nucleic acid probes may be made using only 2 or only 3 of the 4 naturally occurring nucleotide bases, such as leaving out all the “G”s or leaving out all of the “C”s within the population of probes. Sequences lacking either “G”s or “C”s may form very little secondary structure in certain embodiments, and can contribute to more uniform, faster hybridization.
- the nucleic acid probes may contain only A, T, and G; only A, T, and C; only A, C, and G; or only T, C, and G.
- the readout sequences on the nucleic acid probes may be able to bind (e.g., specifically) to corresponding recognition sequences on the primary amplifier nucleic acids.
- the primary amplifier nucleic acid are also able to associate with the target via the nucleic acid probe, with interactions between the readout sequences of the nucleic acid probes and corresponding recognition sequences on the primary amplifier nucleic acids, e.g., complementary binding.
- the recognition sequence may be able to recognize a target readout sequence, but not substantially recognize or bind to other, non-target readout sequence.
- the primary amplifier nucleic acids may also comprise any of a variety of entities able to hybridize a nucleic acid, e.g., DNA, RNA, LNA, and/or PNA, etc., depending on the application. For instance, such entities may form some or all of the recognition sequence.
- the recognition sequence may be substantially complementary to the target readout sequence.
- the sequences may be at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary.
- complementarity is determined on the basis of Watson-Crick nucleotide base pairing.
- the structures of the target readout sequence may include those previously described.
- the recognition sequence may be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 65, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 nucleotides in length.
- the recognition sequence may be no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, no more than 175, no more than 150, no more than 125, no more than 100, be no more than 75, no more than 60, no more than 65, no more than 60, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 20, or no more than 10 nucleotides in length.
- the recognition sequence may have a length of between 10 and 30 nucleotides, between 20 and 40 nucleotides, between 5 and 50 nucleotides, between 10 and 200 nucleotides, or between 25 and 35 nucleotides, between 10 and 300 nucleotides, etc.
- a primary amplifier nucleic acid may also comprise one or more readout sequences able to bind to secondary amplifier nucleic acids, as discussed below.
- a primary amplifier nucleic acid may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9,
- the primary amplifier nucleic acid comprises a recognition sequence at a first end and a plurality of readout sequences at a second end.
- a readout sequence within the primary amplifier nucleic acid may be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 65, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 nucleotides in length.
- the readout sequence may be no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, no more than 175, no more than 150, no more than 125, no more than 100, be no more than 75, no more than 60, no more than 65, no more than 60, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 20, or no more than 10 nucleotides in length.
- the readout sequence may have a length of between 10 and 20 nucleotides, between 10 and 30 nucleotides, between 20 and 40 nucleotides, between 5 and 50 nucleotides, between 10 and 200 nucleotides, or between 25 and 35 nucleotides, between 10 and 300 nucleotides, etc.
- a primary amplifier nucleic acid there may be any number of readout sequences within a primary amplifier nucleic acid. For example, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more readout sequences present within a primary amplifier nucleic acid. If more than one read sequence is present within a primary amplifier nucleic acid, the readout sequences may be the same or different. In some cases, for example, the readout sequences may all be identical.
- the population of primary amplifier nucleic acids may be made using only 2 or only 3 of the 4 naturally occurring nucleotide bases, such as leaving out all the “G”s or leaving out all of the “C”s within the population of nucleic acids. Sequences lacking either “G”s or “C”s may form very little secondary structure in certain embodiments, and can contribute to more uniform, faster hybridization.
- the primary amplifier nucleic acids may contain only A, T, and G; only A, T, and C; only A, C, and G; or only T, C, and G.
- more than one type of primary amplifier nucleic acid may be applied to a sample, e.g., sequentially or simultaneously. For example, there may be at least 2, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 300, at least 1,000, at least 3,000, at least 10,000, or at least 30,000 distinguishable primary amplifier nucleic acids that are applied to a sample.
- the primary amplifier nucleic acids may be added sequentially. However, in some cases, more than one primary amplifier nucleic acid may be added simultaneously.
- the readout sequences on the primary amplifier nucleic acids may be able to bind (e.g., specifically) to corresponding recognition sequences on the secondary amplifier nucleic acids.
- a nucleic acid probe recognizes a target within a biological sample, e.g., a DNA or RNA target
- the secondary amplifier nucleic acids are also able to associate with the target, via the primary amplifier nucleic acids, with interactions between the readout sequences of the primary amplifier nucleic acids and corresponding recognition sequences on the secondary amplifier nucleic acids, e.g., complementary binding.
- the recognition sequence on a secondary amplifier nucleic acid may be able to recognize a readout sequence on a primary amplifier nucleic acid, but not substantially recognize or bind to other, non-target readout sequence.
- the secondary amplifier nucleic acids may also comprise any of a variety of entities able to hybridize a nucleic acid, e.g., DNA, RNA, LNA, and/or PNA, etc., depending on the application. For instance, such entities may form some or all of the recognition sequence.
- the recognition sequence on the secondary amplifier nucleic acid may be substantially complementary to a readout sequence on a primary amplifier nucleic acid.
- the sequences may be at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary.
- the recognition sequence on the secondary amplifier nucleic acid may be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 65, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 nucleotides in length.
- the recognition sequence may be no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, no more than 175, no more than 150, no more than 125, no more than 100, be no more than 75, no more than 60, no more than 65, no more than 60, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 20, or no more than 10 nucleotides in length.
- the recognition sequence may have a length of between 10 and 30 nucleotides, between 20 and 40 nucleotides, between 5 and 50 nucleotides, between 10 and 200 nucleotides, or between 25 and 35 nucleotides, between 10 and 300 nucleotides, etc.
- a secondary amplifier nucleic acid may comprise a signaling entity, and/or may comprise one or more readout sequences able to bind to a signaling entity, as discussed herein.
- a secondary amplifier nucleic acid may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more, 20 or more, 32 or more, 40 or more, 50 or more, 64 or more, 75 or more, 100 or more, 128 or more readout sequences able to bind to a signaling entity.
- the read sequences may be positioned anywhere within the secondary amplifier nucleic acid. If more than one readout sequences is present, the readout sequences may be positioned next to each other, and/or interspersed with other sequences.
- the secondary amplifier nucleic acid comprises a recognition sequence at a first end and a plurality of readout sequences at a second end. This structure may also be the same or different than the structure of the primary amplifier nucleic acid.
- the readout sequence within the secondary amplifier nucleic acid may be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 65, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 nucleotides in length.
- the readout sequence may be no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, no more than 175, no more than 150, no more than 125, no more than 100, be no more than 75, no more than 60, no more than 65, no more than 60, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 20, or no more than 10 nucleotides in length.
- the readout sequence within the secondary amplifier nucleic acid may have a length of between 10 and 20 nucleotides, between 10 and 30 nucleotides, between 20 and 40 nucleotides, between 5 and 50 nucleotides, between 10 and 200 nucleotides, or between 25 and 35 nucleotides, between 10 and 300 nucleotides, etc.
- a secondary amplifier nucleic acid there may be any number of readout sequences within a secondary amplifier nucleic acid. For example, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more readout sequences present within a secondary amplifier nucleic acid. If more than one readout sequence is present within a secondary amplifier nucleic acid, the readout sequences may be the same or different. In some cases, for example, the readout sequences may all be identical. In addition, there may independently be the same or different numbers of readout sequences in the primary and in the secondary amplifier nucleic acids.
- the population of secondary amplifier nucleic acids may be made using only 2 or only 3 of the 4 naturally occurring nucleotide bases, in certain embodiments such as leaving out all the “G”s or leaving out all of the “C”s within the population of nucleic acids. Sequences lacking either “G”s or “C”s may form very little secondary structure in certain embodiments, and can contribute to more uniform, faster hybridization. Thus, in some cases, the secondary amplifier nucleic acids may contain only A, T, and G; only A, T, and C; only A, C, and G; or only T, C, and G.
- more than one type of secondary amplifier nucleic acid may be applied to a sample, e.g., sequentially or simultaneously. For example, there may be at least 2, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 300, at least 1,000, at least 3,000, at least 10,000, or at least 30,000 distinguishable secondary amplifier nucleic acids that are applied to a sample.
- the secondary amplifier nucleic acids may be added sequentially. However, in some cases, more than one secondary amplifier nucleic acid may be added simultaneously.
- this pattern can instead be repeated prior to the signaling entity, e.g., with tertiary amplifier nucleic acids, quaternary nucleic acids, etc., similar to the above discussion.
- the signaling entities may thus be bound to the ending amplifier nucleic acid.
- an encoding nucleic acid probe to which a primary amplifier nucleic acid is bound, to which a secondary amplifier nucleic acid is bound, to which a tertiary amplifier nucleic acid is bound, to which a signaling entity is bound
- a target may be bound an encoding nucleic acid probe, to which a primary amplifier nucleic acid is bound, to which a secondary amplifier nucleic acid is bound, to which a tertiary amplifier nucleic acid is bound, to which a quaternary amplifier nucleic acid is bound, to which a signaling entity is bound, etc.
- the ending amplifier nucleic acid need not necessarily be the secondary amplifier nucleic acid in all embodiments.
- FIG. 5A-5E show the creation of a saturatable system.
- Fig. 5A shows an example of an encoding nucleic acid probe, where an encoding nucleic acid probe 15 has bound to a target RNA.
- Fig. 5B shows a primary amplifier nucleic acid being used, in accordance with certain embodiments.
- Fig. 5C shows a secondary amplifier nucleic acid that may be bound to the primary amplifier nucleic acid.
- Fig. 5D shows that a plurality of signaling entities has been bound to the readout sequences of the secondary amplifier nucleic acids.
- Fig. 5E shows that if no amplification is applied, the nucleic acid probe may be exposed to a suitable secondary nucleic acid probe containing a signaling entity.
- primer sequences may be present, e.g., to facilitate enzymatic amplification.
- primer sequences suitable for applications such as amplification (e.g., using PCR or other suitable techniques). Many such primer sequences are available commercially.
- sequences that may be present within a primary or encoding nucleic acid probe include, but are not limited to promoter sequences, operons, identification sequences, nonsense sequences, or the like.
- a primer is a single- stranded or partially double-stranded nucleic acid (e.g., DNA) that serves as a starting point for nucleic acid synthesis, allowing polymerase enzymes such as nucleic acid polymerase to extend the primer and replicate the complementary strand.
- a primer is (e.g., is designed to be) complementary to and to hybridize to a target nucleic acid.
- a primer is a synthetic primer.
- a primer is a non-naturally-occurring primer.
- a primer typically has a length of 10 to 50 nucleotides.
- a primer may have a length of 10 to 40, 10 to 30, 10 to 20, 25 to 50, 15 to 40, 15 to 30, 20 to 50, 20 to 40, or 20 to 30 nucleotides. In some embodiments, a primer has a length of 18 to 24 nucleotides.
- certain embodiments use code spaces that encode various binding events, and optionally can use error detection and/or correction to determine the binding of nucleic acid probes to their targets.
- a population of nucleic acid probes may contain certain “readout sequences” which can bind certain amplifier nucleic acids, as discussed above, and the locations of the nucleic acid probes or targets can be determined within the sample using signaling entities associated with the amplifier nucleic acids, for example, within a certain code space, e.g., as discussed herein. See also Int. Pat. Apl. Pub. Nos. WO 2016/018960 and WO 2016/018963, each incorporated herein by reference in its entirety.
- a population of readout sequences within the nucleic acid probes may be combined in various combinations, e.g., such that a relatively small number of readout sequences may be used to determine a relatively large number of different nucleic acid probes, as discussed herein.
- a population of nucleic acid probes may each contain a certain number of readout sequences, some of which are shared between different nucleic acid probes such that the total population of nucleic acid probes may contain a certain number of readout sequences.
- a population of nucleic acid probes may have any suitable number of readout sequences. For example, a population of nucleic acid probes may have 1, 2, 3, 4, 5,
- a population of nucleic acid probes may, in total, have 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more,
- the population of nucleic acid probes may have no more than 100, no more than 80, no more than 64, no more than 60, no more than 50, no more than 40, no more than 32, no more than 24, no more than 20, no more than 16, no more than 15, no more than 14, no more than 13, no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, or no more than two readout sequences present. Combinations of any of these are also possible, e.g., a population of nucleic acid probes may comprise between 10 and 15 readout sequences in total.
- the total number of readout sequences within the population may be no greater than 4. It should be understood that although 4 readout sequences are used in this example for ease of explanation, in other embodiments, larger numbers of nucleic acid probes may be realized, for example, using 5, 8, 10, 16, 32, etc.
- each of the nucleic acid probes or each group of the nucleic acid probes contain two different readout sequences, then by using 4 such read sequences (A, B, C, and D), up to 6 probes or 6 groups of probes may be separately identified.
- the ordering of readout sequences on a nucleic acid probe or a group of nucleic acid probes is not essential, i.e., “AB” and “BA” may be treated as being synonymous (although in other embodiments, the ordering of read sequences may be essential and “AB” and “BA” may not necessarily be synonymous).
- probes or 10 groups of probes may be separately identified (e.g., AB, AC, AD, AE, BC, BD, BE, CD, CE, DE).
- each group of probes bind to a nucleic acid target.
- the readout sequences and/or the pattern of binding of nucleic acid probes within a sample may be used to define an error-detecting and/or an error-correcting code, for example, to reduce or prevent misidentification or errors of the nucleic acids.
- binding e.g., as determined using a signaling entity
- the location may be identified with a “1”; conversely, if no binding is indicated, then the location may be identified with a “0” (or vice versa, in some cases).
- Multiple rounds of binding determinations e.g., using different readout probes complementary to readout sequences, can then be used to create a “codeword,” e.g., for that spatial location.
- the codeword may be subjected to error detection and/or correction.
- the codewords may be organized such that, if no match is found for a given set of readout sequences or binding pattern of nucleic acid probes, then the match may be identified as an error, and optionally, error correction may be applied sequences to determine the correct target for the nucleic acid probes.
- the codewords may have fewer “letters” or positions than the total number of nucleic acids encoded by the codewords, e.g. where each codeword encodes a different nucleic acid.
- Such error-detecting and/or the error-correction code may take a variety of forms.
- a variety of such codes have previously been developed in other contexts such as the telecommunications industry, such as Golay codes or Hamming codes.
- the readout sequences or binding patterns of the nucleic acid probes are assigned such that not every possible combination is assigned.
- nucleic acid probes or a group of nucleic acid probes contains 2 readout sequences
- up to 6 nucleic acid probes or 6 groups of nucleic acid probes e.g., such that each group of nucleic acid probes binds to a nucleic acid target
- the number of nucleic acid probes or the number of groups of nucleic acid probes used may be less than 6.
- nucleic acid probes or the number of groups of nucleic acid probes that are used may be any number more or less than (fc).
- these may be randomly assigned, or assigned in specific ways to increase the ability to detect and/or correct errors.
- the number of rounds may be arbitrarily chosen. If in each round, each target can give two possible outcomes, such as being detected or not being detected, up to 2" different targets may be possible for n rounds of probes, but the number of targets that are actually used may be any number less than 2". In another example, if in each round, each target can give more than two possible outcomes, such as being detected in different color channels, more than 2" (e.g. 3", 4", ...) different targets may be possible for n rounds of probes. In some cases, the number of targets that are actually used may be any number less than this number. In addition, these may be randomly assigned, or assigned in specific ways to increase the ability to detect and/or correct errors.
- the codewords may be used to define various code spaces. Each nucleic acid target is associated with a codeword.
- the codewords may be assigned within a code space such that the assignments are separated by a Hamming distance, which measures the number of incorrect “reads” in a given pattern that cause the codeword or the associated nucleic acid target to be misinterpreted as a different valid codeword or nucleic acid target.
- the Hamming distance may be at least 2, at least 3, at least 4, at least 5, at least 6, or the like.
- the assignments may be formed as a Hamming code, for instance, a Hamming(7, 4) code, a Hamming(15, 11) code, a Hamming(31, 26) code, a Hamming(63, 57) code, a Hamming(127, 120) code, etc.
- the assignments may form a SECDED code, e.g., a SECDED(8,4) code, a SECDED(16,4) code, a SCEDED(16, 11) code, a SCEDED(22, 16) code, a SCEDED(39, 32) code, a SCEDED(72, 64) code, etc.
- the assignments may form an extended binary Golay code, a perfect binary Golay code, or a ternary Golay code.
- the assignments may represent a subset of the possible values taken from any of the codes described above.
- an error-correcting code may be formed by using only binary words that contain a fixed or constant number of “1” bits (or “0” bits) to encode the targets.
- the code space may only include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, etc. “1” bits (or “0” bits), e.g., all of the codes have the same number of “1” bits or “0” bits, etc.
- the assignments may represent a subset of the possible values taken from codes described above for the purpose of addressing asymmetric readout errors.
- a code in which the number of “1” bits may be fixed for all used binary words may eliminate the biased measurement of words with different numbers of “l”s when the rate at which “0” bits are measured as “l”s or “1” bits are measured as “0”s are different.
- the codeword may be compared to the valid nucleic acid codewords. If a match is found, then the nucleic acid target can be identified or determined. If no match is found, then an error in the reading of the codeword may be identified. In some cases, error correction can also be applied to determine the correct codeword, and thus resulting in the correct identity of the nucleic acid target. In some cases, the codewords may be selected such that, assuming that there is only one error present, only one possible correct codeword is available, and thus, only one correct identity of the nucleic acid target is possible.
- this may also be generalized to larger codeword spacings or Hamming distances; for instance, the codewords may be selected such that if two, three, or four errors are present (or more in some cases), only one possible correct codeword is available, and thus, only one correct identity of the nucleic acid targets is possible.
- the error-correcting code may be a binary error-correcting code, or it may be based on other numbering systems, e.g., ternary or quaternary error-correcting codes.
- more than one type of signaling entity may be used and assigned to different numbers within the error-correcting code.
- a first signaling entity (or more than one signaling entity, in some cases) may be assigned as “1” and a second signaling entity (or more than one signaling entity, in some cases) may be assigned as “2” (with “0” indicating no signaling entity present), and the codewords distributed to define a ternary error-correcting code.
- a third signaling entity may additionally be assigned as “3” to make a quaternary error-correcting code, etc.
- nucleic acid targets in a sample are each assigned with a codeword.
- these codewords could be chosen from one of the codespaces as describe herein.
- the codewords form an error-detecting and/or error correcting code.
- the sample may be subjected to hybridization to a population of primary or encoding nucleic acid probes in some cases.
- Some or all of the primary or encoding probe may comprise a target sequence that can bind to one of the nucleic acid targets and/or may also comprises one or more readout sequences.
- the readout sequences on the collection of primary or encoding probes that bind to each nucleic acid target may form a unique codeword that corresponds to the codeword assigned to the nucleic acid target.
- the sample are then subject to one or more rounds of hybridization with readout probes.
- the readout probes may be able to bind to a readout sequence and/or may be associated with a signaling entity.
- the collection of readout sequences may be associated with a nucleic acid target, and hence the codeword assigned to the nucleic acid target can then be identified, e.g., through the binding of readout probes.
- multi-color imaging can be used in each round to allow simultaneous imaging and determination of multiple readout probes associated with different signaling entities.
- the positions of the nucleic acid targets are determined. In some cases, at least 50, at least 100, at least 500, at least 1000, at least 5000, or at least 10,000 nucleic acid targets are determined this way.
- the target nucleic acids are genomic loci. In some cases, the target nucleic acids are genomic loci and/or nascent RNA transcripts. In some cases, the positions of the genomic loci are used to determine the three- dimensional organization of chromatin or the three-dimensional organization of the genome in the cell.
- primary amplifier nucleic acids and/or secondary amplifier nucleic acids and/or tertiary amplifier nucleic acids and/or quaternary amplifier nucleic acids are used to amplify the signal from each readout sequence.
- adaptors are used as described below.
- a plurality of adapters may be used to facilitate detection of targets within a sample.
- Such adapters may be useful, for example, in allowing a relatively small number of distinguishable signaling entities to be used, while still allowing for relatively large numbers of targets to be determined in a sample.
- At least 3, at least 4, at least 7, at least 8, at least 12, at least 14, at least 15, at least 16, at least 20, at least 22, at least 30, at least 31, at least 32, at least 50, at least 63, at least 64, at least 72, at least 75, at least 100, at least 127, at least 128, at least 140, at least 255, at least 256, at least 500, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 4,000, at least 5,000, at least 7,500, at least 10,000, at least 12,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 40,000, at least 50,000, at least 75,000, or at least 100,000, etc. targets in a sample may be determined, while using a smaller number of signaling entities, for example, no more than 20, no more than 15, no more than 10, no more than 5, no more than 4, no more than 3, or no more than 2 signaling entities.
- a plurality of adaptors may be used.
- the adaptors may comprise a first portion substantially complementary to one or more readout sequences on a nucleic acid probe (e.g., a primary nucleic acid probe), and a second portion comprising one or more identification sequences.
- the adaptor sequences are thus able to bind to specific nucleic acid probes that may be bound to a target in the sample.
- the identification sequences are then available for binding, e.g., via readout probes or secondary nucleic acid probes, such as those discussed herein.
- the adaptor may be present between the primary nucleic acid probes and the secondary nucleic acid probes.
- Fig. 24A One non-limiting example of this is shown in Fig. 24A.
- the adaptors may be chosen to allow a relatively small number of signaling entities to be used, as noted above.
- the identification sequences may act as readout sequences that the secondary nucleic acid probes are able to bind to.
- a relatively small number of secondary nucleic acid probes e.g., containing a signaling entity and a sequence substantially complementary to one of the identification sequences, may be used, and the signaling entities determined, e.g., as discussed herein.
- the secondary nucleic acid probes may then be removed and/or deactivated, e.g., as described herein, before the next round of detection.
- the next and subsequent rounds may use the same or different signaling entities, e.g., on secondary nucleic acid probes containing sequences substantially complementary to different identification sequences.
- the adapters used in the previous round may be deactivated in some fashion.
- blocking nucleic acid probes may be added that contain sequences substantially complementary to the previous identification sequences, such that they are able to bind to the previous adaptors, but since they are not generally detectable without the presence of a signaling entity. Accordingly, in subsequent rounds of detection, signals due to prior rounds may be minimized.
- relatively large numbers of identification sequences may be determined using no more than a relatively small number of signaling entities. For instance, at least 3, at least 4, at least 7, at least 8, at least 12, at least 14, at least 15, at least 16, at least 20, at least 22, at least 30, at least 31, at least 32, at least 50, at least 63, at least 64, at least 72, at least 75, at least 100, at least 127, at least 128, at least 140, at least 255, at least 256, at least 500, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 4,000, at least 5,000, at least 7,500, at least 10,000, at least 12,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 40,000, at least 50,000, at least 75,000, or at least 100,000, etc. identification sequences may be determined using no more than 20, no more than 15, no more than 10, no more than 5, no more than 4, no more than 3, or no more than 2 signaling entities
- the identification sequences may be of any length. If more than one identification sequence is used, the identification sequences may independently have the same or different lengths. For instance, the identification sequence may be at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 65, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 nucleotides in length.
- the identification sequence may be no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, no more than 175, no more than 150, no more than 125, no more than 100, be no more than 75, no more than 60, no more than 65, no more than 60, no more than 55, no more than 50, no more than 45, no more than 40, no more than 35, no more than 30, no more than 20, or no more than 10 nucleotides in length.
- the identification sequence may have a length of between 10 and 30 nucleotides, between 20 and 40 nucleotides, between 5 and 50 nucleotides, between 10 and 200 nucleotides, or between 25 and 35 nucleotides, between 10 and 300 nucleotides, etc.
- the identification sequence may be arbitrary or random in some embodiments. In certain cases, the identification sequences are chosen so as to reduce or minimize homology with other components of the cell or other sample, e.g., such that the identification sequences do not themselves bind to or hybridize with other nucleic acids suspected of being within the cell or other sample. In some cases, the homology may be less than 10%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.
- the identification sequences may be selected such that they do not exhibit specific binding towards each other and/or towards the genome or other nucleic acids suspected of being present in the sample, such as readout sequences. For instance, a population of identification sequences may be “blasted” or tested for specific binding or complementarity. In some case, the identification sequences may exhibit no specific binding towards each other, and/or such that none of the identification sequences in the population of identification sequences has a complementary of more than 5, 6, 7, 8, 9, 10, etc. nucleotides to another sequence within the population of identification sequences, and/or within a population of readout sequences.
- a sample is first subject to hybridization to a population of primary or encoding nucleic acid probes.
- One or more of the primary or encoding probe comprises a target sequence that can bind to one of the nucleic acid targets and may also comprises one or more readout sequences.
- the sample are then subject to multiple rounds of hybridization with adaptor probes and readout probes.
- the adaptor probes may comprise a sequence that can bind to a readout sequence and also comprises one or more identification sequence.
- the readout probe may be able to bind to an identification sequence and is also associated with a signaling entity.
- multi-color imaging can be used in each round to allow simultaneous imaging and determination of multiple readout probes associated with different signaling entities.
- signaling entities are determined, e.g., by imaging, to determine nucleic acid probes and/or to create codewords. Examples of signaling entities include those discussed herein.
- signaling entities within a sample may be determined, e.g., spatially, using a variety of techniques.
- the signaling entities may be fluorescent, and techniques for determining fluorescence within a sample, such as fluorescence microscopy or confocal microscopy, may be used to spatially identify the positions of signaling entities within a cell.
- the positions of entities within the sample may be determined in two or even three dimensions.
- more than one signaling entity may be determined at a time (e.g., signaling entities with different colors or emissions), and/or sequentially.
- a confidence level for an identified target may be determined.
- the confidence level may be determined using a ratio of the number of exact matches to the number of matches having one or more one-bit errors. In some cases, only matches having a confidence ratio greater than a certain value may be used.
- matches may be accepted only if the confidence ratio for the match is greater than about 0.01, greater than about 0.03, greater than about 0.05, greater than about 0.1, greater than about 0.3, greater than about 0.5, greater than about 1, greater than about 3, greater than about 5, greater than about 10, greater than about 30, greater than about 50, greater than about 100, greater than about 300, greater than about 500, greater than about 1000, or any other suitable value.
- matches may be accepted only if the confidence ratio for the identified target is greater than an internal standard or false positive control by about 0.01, about 0.03, about 0.05, about 0.1, about 0.3, about 0.5, about 1, about 3, about 5, about 10, about 30, about 50, about 100, about 300, about 500, about 1000, or any other suitable value
- the spatial positions of the entities may be determined at relatively high resolutions.
- the positions may be determined at spatial resolutions of better than about 100 micrometers, better than about 30 micrometers, better than about 10 micrometers, better than about 3 micrometers, better than about 1 micrometer, better than about 800 nm, better than about 600 nm, better than about 500 nm, better than about 400 nm, better than about 300 nm, better than about 200 nm, better than about 100 nm, better than about 90 nm, better than about 80 nm, better than about 70 nm, better than about 60 nm, better than about 50 nm, better than about 40 nm, better than about 30 nm, better than about 20 nm, or better than about 10 nm, etc.
- the spatial positions of entities optically e.g., using fluorescence microscopy. More than one color can be used in some embodiments.
- the spatial positions may be determined at super resolutions, or at resolutions better than the wavelength of light or the diffraction limit.
- Non limiting examples include STORM (stochastic optical reconstruction microscopy), STED (stimulated emission depletion microscopy), NSOM (Near-field Scanning Optical Microscopy), 4Pi microscopy, SIM (Structured Illumination Microscopy), SMI (Spatially Modulated Illumination) microscopy, RESOLFT (Reversible Saturable Optically Linear Fluorescence Transition Microscopy), GSD (Ground State Depletion Microscopy), SSIM (Saturated Structured-Illumination Microscopy), SPDM (Spectral Precision Distance Microscopy), Photo-Activated localization Microscopy (PALM), Fluorescence Photoactivation Localization Microscopy (FPALM), LIMON (3D Light Microscopical Nanosizing Microscopy), Super-resolution optical fluctuation imaging (SOFI), Expansion Microscopy, or the like.
- the sample may be imaged with a high numerical aperture, oil immersion objective with 100X magnification and light collected on an electron-multiplying CCD camera.
- the sample could be imaged with a high numerical aperture, oil immersion lens with 40X magnification and light collected with a wide-field scientific CMOS camera.
- a single field of view may correspond to no less than l x l microns, 10 x 10 microns, 40 x 40 microns, 80 x 80 microns, 120 x 120 microns, 240 x 240 microns, 340 x 340 microns, or 500 x 500 microns, etc.
- a single camera pixel may correspond, in some embodiments, to regions of the sample of no less than 10x10 nm, 20x20 nm, 40x40 nm, 80x80 nm, 120x120 nm, 160x160 nm, 240x240 nm, or 300x300 nm, etc.
- the sample may be imaged with a low numerical aperture, air lens with 10X magnification and light collected with a sCMOS camera.
- the sample may be optically sectioned by illuminating it via a single or multiple scanned diffraction limited foci generated either by scanning mirrors or a spinning disk and the collected passed through a single or multiple pinholes.
- the sample may also be illuminated via a thin sheet of light generated via any one of multiple methods known to those versed in the art.
- the sample may be illuminated by single Gaussian mode laser lines.
- the illumination profiled may be flattened by passing these laser lines through a multimode fiber that is vibrated via piezo-electric or other mechanical means.
- the illumination profile may be flattened by passing single-mode, Gaussian beams through a variety of refractive beam shapers, such as the piShaper or a series of stacked Powell lenses.
- the Gaussian beams may be passed through a variety of different diffusing elements, such as ground glass or engineered diffusers, which may be spun in some cases at high speeds to remove residual laser speckle.
- laser illumination may be passed through a series of lenslet arrays to produce overlapping images of the illumination that approximate a flat illumination field.
- the centroids of the spatial positions of the entities may be determined.
- a centroid of a signaling entity may be determined within an image or series of images using image analysis algorithms known to those of ordinary skill in the art.
- the algorithms may be selected to determine non-overlapping single emitters and/or partially overlapping single emitters in a sample.
- suitable techniques include a maximum likelihood algorithm, a least squares algorithm, a Bayesian algorithm, a compressed sensing algorithm, or the like. Combinations of these techniques may also be used in some cases.
- one or more signaling entities may be determined.
- a signaling entity may be bound to the readout probe or the recognition entities on the secondary amplifier nucleic acids (or other ending amplifier nucleic acid).
- Non-limiting examples of signaling entities include fluorescent entities (fluorophores) or phosphorescent entities, e.g., as discussed herein.
- the signaling entities may then be determined, e.g., to determine the nucleic acid probes or the targets.
- the determination may be spatial, e.g., in two or three dimensions.
- the determination may be quantitative, e.g., the amount or concentration of signaling entity and/or of a target may be determined.
- the signaling entities may be attached to the secondary amplifier nucleic acid (or other ending amplifier nucleic acid).
- the signaling entities may be attached to the secondary amplifier nucleic acid (or other ending amplifier nucleic acid) before or after association of the secondary amplifier nucleic acid to targets within the sample.
- the signaling entities may be attached to the secondary amplifier nucleic acid initially, or after the secondary amplifier nucleic acids have been applied to a sample. In some cases, the signaling entities are added, then reacted to attach them to the amplifier nucleic acids.
- the signaling entities may be attached to a nucleotide sequence via a bond that can be cleaved to release the signaling entity.
- the bond may be a cleavable bond, such as a disulfide bond or a photocleavable bond. Examples of photocleavable bonds are discussed in detail herein. In some cases, such bonds may be cleaved, for example, upon exposure to reducing agents or light (e.g., ultraviolet light). See below for additional details.
- the signaling entity is deactivated by photobleaching. Other examples of systems and methods for inactivating and/or removing the signaling entity are discussed in more detail herein.
- the use of primary and secondary amplifier nucleic acids may be used to create a maximum number of signaling entities that can be bound to a given nucleic acid probe. For instance, there may be a maximum number of signaling entities that are able to bind to a nucleic acid probe, e.g., due to a maximum number of readout probes with signaling entities that are able to bind to a finite number of secondary amplifier nucleic acids, due to a maximum number of secondary amplifier nucleic acids that are able to bind to a finite number of primary amplifier nucleic acids, and/or due to a maximum number of primary amplifier nucleic acids that are able to bind to the finite number of read sequences on the nucleic acid probes. While each potential location need not actually be filled with a signaling entity, this structure suggests that there is a saturation limit of signaling entities, beyond which any additional signaling entities that may happen to be present are unable to associate with a nucleic acid probe or its target.
- certain embodiments are generally directed to systems and methods of amplifying a signal indicating a nucleic acid probe or its target that are saturatable, i.e., such that there is an upper, saturation limit of how many signaling entities can associate with the nucleic acid probe or its target.
- the upper limit of signaling entities may be at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 250, at least 300, at least 400, at least 500, etc.
- the upper limit may be less than 500, less than 400, less than 300, less than 250, less than 200, less than 175, less than 150, less than 125, less than 100, less than 75, less than 50, less than 40, less than 30, less than 25, less than 20, less than 15, less than 10, less than 5, etc.
- the upper limit may be determined as the maximum number of signaling entities that can bind to a secondary amplifier nucleic acid, multiplied by the maximum number of secondary amplifier nucleic acids that can bind to a primary amplifier nucleic acid, multiplied by the maximum number of primary amplifier nucleic acids that can bind to a nucleic acid probe that binds to a target.
- the average number of signaling entities actually bound to a nucleic acid probe or its target need not actually be the same as its upper limit, i.e., the signaling entities may not actually be at full saturation (although they can be).
- the amount of saturation (or the number of signaling entities bound, relative to the maximum number that can bind) may be less than 97%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, etc., and/or at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, etc. In some cases, allowing more time for binding to occur and/or increasing the concentration of reagents may increase the amount of saturation.
- the binding events distributed within a sample may present substantially uniform sizes and/or brightnesses, in contrast to uncontrolled amplifications, such as those discussed above.
- the secondary amplifier nucleic acids cannot be found greater than a fixed distance from the nucleic acid probe or its target, which may limit the “spot size” or diameter of fluorescence from the signaling entities, indicating binding.
- At least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the binding events may exhibit substantially the same brightnesses, sizes (e.g., apparent diameters), colors, or the like, which may make it easier to distinguish binding events from other events, such as nonspecific binding, noise, or the like.
- the signaling entity may be inactivated in some cases.
- a first secondary nucleic acid probe or readout probe that can associate with a signaling entity e.g., using amplifier nucleic acids
- a sample that can recognize a first readout sequence e.g., on the primary or encoding nucleic acid probe
- the signaling entity can be inactivated before a second secondary nucleic acid probe or readout probe is applied to the sample, e.g., that can associate with a signaling entity (e.g., using amplifier nucleic acids).
- the same or different techniques may be used to inactivate the signaling entities, and some or all of the multiple signaling entities may be inactivated, e.g., sequentially or simultaneously.
- Inactivation may be caused by removal of the signaling entity (e.g., from the sample, or from the nucleic acid probe, etc.), and/or by chemically altering the signaling entity in some fashion (e.g., by photobleaching the signaling entity, bleaching or chemically altering the structure of the signaling entity, for example, by reduction, etc.).
- removal of the signaling entity e.g., from the sample, or from the nucleic acid probe, etc.
- chemically altering the signaling entity in some fashion e.g., by photobleaching the signaling entity, bleaching or chemically altering the structure of the signaling entity, for example, by reduction, etc.
- a fluorescent signaling entity may be inactivated by chemical or optical techniques such as oxidation, photobleaching, chemically bleaching, stringent washing or enzymatic digestion or reaction by exposure to an enzyme, dissociating the signaling entity from other components (e.g., a probe), chemical reaction of the signaling entity (e.g., to a reactant able to alter the structure of the signaling entity) or the like.
- chemical or optical techniques such as oxidation, photobleaching, chemically bleaching, stringent washing or enzymatic digestion or reaction by exposure to an enzyme, dissociating the signaling entity from other components (e.g., a probe), chemical reaction of the signaling entity (e.g., to a reactant able to alter the structure of the signaling entity) or the like.
- bleaching may occur by exposure to oxygen, reducing agents, or the signaling entity could be chemically cleaved from the nucleic acid probe and washed away via fluid flow.
- various nucleic acid probes may be associated with one or more signaling entities, e.g., using amplifier nucleic acids as discussed herein. If more than one nucleic acid probe (or secondary nucleic acid probes or readout probes) is used, the signaling entities may each be the same or different.
- a signaling entity is any entity able to emit light. For instance, in one embodiment, the signaling entity is fluorescent. In other embodiments, the signaling entity may be phosphorescent, radioactive, absorptive, etc. In some cases, the signaling entity is any entity that can be determined within a sample at relatively high resolutions, e.g., at resolutions better than the wavelength of visible light or the diffraction limit.
- the signaling entity may be, for example, a dye, a small molecule, a peptide or protein, or the like.
- the signaling entity may be a single molecule in some cases. If multiple secondary nucleic acid probes or readout probes are used, the nucleic acid probes may associate with the same or different signaling entities.
- Non-limiting examples of signaling entities include fluorescent entities (fluorophores) or phosphorescent entities, for example, cyanine dyes (e.g., Cy2, Cy3, Cy3B, Cy5, Cy5.5, Cy7, etc.), Alexa Fluor dyes, Atto dyes, photoswitchable dyes, photoactivatable dyes, fluorescent dyes, metal nanoparticles, semiconductor nanoparticles or “quantum dots,”
- fluorescent entities fluorophores
- phosphorescent entities for example, cyanine dyes (e.g., Cy2, Cy3, Cy3B, Cy5, Cy5.5, Cy7, etc.), Alexa Fluor dyes, Atto dyes, photoswitchable dyes, photoactivatable dyes, fluorescent dyes, metal nanoparticles, semiconductor nanoparticles or “quantum dots,”
- the signaling entity may be attached to an oligonucleotide sequence via a bond that can be cleaved to release the signaling entity.
- a fluorophore may be conjugated to an oligonucleotide via a cleavable bond, such as a photocleavable bond.
- Non-limiting examples of photocleavable bonds include, but are not limited to, l-(2-nitrophenyl)ethyl, 2-nitrobenzyl, biotin phosphoramidite, acrylic phosphoramidite, diethylaminocoumarin, l-(4,5-dimethoxy-2-nitrophenyl)ethyl, cyclo- dodecyl (dimethoxy-2-nitrophenyl)ethyl, 4-aminomethyl-3-nitrobenzyl, (4-nitro-3-(l- chlorocarbonyloxyethyl)phenyl)methyl-S-acetylthioic acid ester, (4-nitro-3-(l- thlorocarbonyloxyethyl)phenyl)methyl-3-(2-pyridyldithiopropionic acid) ester, 3-(4,4’- dimethoxytrityl)-l-(2-nitrophenyl)-propane-l,3-diol-[2- cyano
- the fluorophore may be conjugated to an oligonucleotide via a disulfide bond.
- the disulfide bond may be cleaved by a variety of reducing agents such as, but not limited to, dithiothreitol, dithioerythritol, beta- mercaptoethanol, sodium borohydride, thioredoxin, glutaredoxin, trypsinogen, hydrazine, diisobutylaluminum hydride, oxalic acid, formic acid, ascorbic acid, phosphorous acid, tin chloride, glutathione, thioglycolate, 2,3-dimercaptopropanol, 2-mercaptoethylamine, 2- aminoethanol, tris(2-carboxyethyl)phosphine, bis(2-mercaptoethyl) sulfone, N,N’-dimethyl- N,N’-bis(mercapto
- the fluorophore may be conjugated to an oligonucleotide via one or more phosphorothioate modified nucleotides in which the sulfur modification replaces the bridging and/or non-bridging oxygen.
- the fluorophore may be cleaved from the oligonucleotide, in certain embodiments, via addition of compounds such as but not limited to iodoethanol, iodine mixed in ethanol, silver nitrate, or mercury chloride.
- the signaling entity may be chemically inactivated through reduction or oxidation.
- a chromophore such as Cy5 or Cy7 may be reduced using sodium borohydride to a stable, non-fluorescence state.
- a fluorophore may be conjugated to an oligonucleotide via an azo bond, and the azo bond may be cleaved with 2-[(2-N-arylamino)phenylazo]pyridine.
- a fluorophore may be conjugated to an oligonucleotide via a suitable nucleic acid segment that can be cleaved upon suitable exposure to DNAse, e.g., an exodeoxyribonuclease or an endodeoxyribonuclease. Examples include, but are not limited to, deoxyribonuclease I or deoxyribonuclease II.
- the cleavage may occur via a restriction endonuclease.
- Non-limiting examples of potentially suitable restriction endonucleases include BamHI, Bsrl, Notl, Xmal, PspAI, Dpnl, Mbol, Mnll, Eco57I, Ksp632I, Dralll, Ahall, Smal, Mlul, Hpal, Apal, Bell, BstEII, Taql, EcoRI, Sacl, Hindll, Haell, Drall, Tsp509I, Sau3AI, Pad, etc. Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially.
- a fluorophore may be conjugated to biotin, and the oligonucleotide conjugated to avidin or streptavidin.
- the probes may be removed using corresponding “toe-hold-probes,” which comprise the same sequence as the secondary or readout probe, as well as an extra number of bases of homology to the primary or encoding probes (e.g., 1-20 extra bases, for example, 5 extra bases). These probes may remove the labeled secondary or readout probe through a strand-displacement interaction.
- the oligonucleotide may be, for example, a primary nucleic acid probe, an encoding nucleic acid probe, a readout probe, a primary or secondary (or other) amplifier nucleic acid, such as those discussed herein.
- the term “light” generally refers to electromagnetic radiation, having any suitable wavelength (or equivalently, frequency).
- the light may include wavelengths in the optical or visual range (for example, having a wavelength of between about 400 nm and about 700 nm, i.e., “visible light”), infrared wavelengths (for example, having a wavelength of between about 300 micrometers and 700 nm), ultraviolet wavelengths (for example, having a wavelength of between about 400 nm and about 10 nm), or the like.
- visible light for example, having a wavelength of between about 400 nm and about 700 nm
- infrared wavelengths for example, having a wavelength of between about 300 micrometers and 700 nm
- ultraviolet wavelengths for example, having a wavelength of between about 400 nm and about 10 nm
- more than one entity may be used, i.e., entities that are chemically different or distinct, for example, structurally. However, in other cases, the entities may be chemically identical or at least substantially chemically identical.
- the signaling entity is “switchable,” i.e., the entity can be switched between two or more states, at least one of which emits light having a desired wavelength. In the other state(s), the entity may emit no light, or emit light at a different wavelength. For instance, an entity may be “activated” to a first state able to produce light having a desired wavelength, and “deactivated” to a second state not able to emit light of the same wavelength. An entity is “photoactivatable” if it can be activated by incident light of a suitable wavelength.
- Cy5 or Alexa 647 can be switched between a fluorescent and a dark state in a controlled and reversible manner by light of different wavelengths, i.e., 633 nm (or 642nm, 647nm, 656 nm) red light can switch or deactivate Cy5 or Alexa 647 to a stable dark state, while 405 nm green light can switch or activate the Cy5 or Alexa 647 back to the fluorescent state.
- the entity can be reversibly switched between the two or more states, e.g., upon exposure to the proper stimuli.
- a first stimuli e.g., a first wavelength of light
- a second stimuli e.g., a second wavelength of light
- Any suitable method may be used to activate the entity.
- incident light of a suitable wavelength may be used to activate the entity to emit light, i.e., the entity is “photoswitchable.”
- the photo switchable entity can be switched between different light-emitting or non-emitting states by incident light, e.g., of different wavelengths.
- the light may be monochromatic (e.g., produced using a laser) or polychromatic.
- the entity may be activated upon stimulation by electric field and/or magnetic field.
- the entity may be activated upon exposure to a suitable chemical environment, e.g., by adjusting the pH, or inducing a reversible chemical reaction involving the entity, etc.
- any suitable method may be used to deactivate the entity, and the methods of activating and deactivating the entity need not be the same.
- the entity may be deactivated upon exposure to incident light of a suitable wavelength, or the entity may be deactivated by waiting a sufficient time.
- a “switchable” entity can be identified by one of ordinary skill in the art by determining conditions under which an entity in a first state can emit light when exposed to an excitation wavelength, switching the entity from the first state to the second state, e.g., upon exposure to light of a switching wavelength, then showing that the entity, while in the second state can no longer emit light (or emits light at a much reduced intensity) when exposed to the excitation wavelength.
- a switchable entity may be switched upon exposure to light.
- the light used to activate the switchable entity may come from an external source, e.g., a light source such as a laser light source, another light-emitting entity proximate the switchable entity, etc.
- the second, light emitting entity in some cases, may be a fluorescent entity, and in certain embodiments, the second, light-emitting entity may itself also be a switchable entity.
- the switchable entity includes a first, light-emitting portion (e.g., a fluorophore), and a second portion that activates or “switches” the first portion. For example, upon exposure to light, the second portion of the switchable entity may activate the first portion, causing the first portion to emit light.
- activator portions include, but are not limited to, Alexa Fluor 405 (Invitrogen), Alexa Fluor 488 (Invitrogen), Cy2 (GE Healthcare), Cy3 (GE Healthcare), Cy3B (GE Healthcare), Cy3.5 (GE Healthcare), or other suitable dyes.
- light-emitting portions include, but are not limited to, Cy3B (GE Healthcare), Cy5, Cy5.5 (GE Healthcare), Cy7 (GE Healthcare), Alexa Fluor 647 (Invitrogen), Alexa Fluor 680 (Invitrogen), Alexa Fluor 700 (Invitrogen), Alexa Fluor 750 (Invitrogen), Alexa Fluor 790 (Invitrogen), DiD, DiR, YOYO-3 (Invitrogen), YO-PRO-3 (Invitrogen), TOT-3 (Invitrogen), TO-PRO-3 (Invitrogen) or other suitable dyes. See, e.g., U.S. Pat. No. 7,838,302, incorporated herein by reference in its entirety.
- the first, light-emitting portion can subsequently be deactivated by any suitable technique (e.g., by directing 647 nm red light to the Cy5 portion of the molecule).
- a plurality of nucleic acid probes are used that have different sequences, and the distribution of each of the nucleic acid probes is sequentially analyzed and used to create “codewords” for each location, based on the binding patterns of each of the nucleic acid probes.
- codewords By selecting nucleic acid probes that define a suitable code space, apparent errors in the observed binding patterns can be identified, and/or discarded and/or corrected to identify the correct codeword, and thus the correct target of the nucleic acid probes within the sample.
- This error-robustness and error-correction system was first introduced for multiplexed error-robust fluorescence in situ hybridization (MERFISH), and has also been subsequently used in various related techniques. See, e.g., Int. Pat. Apl. Pub. Nos. WO 2016/018960 and WO 2016/018963, each incorporated herein by reference in its entirety.
- codewords may be based on the binding (or non-binding) of the plurality of readout probes that can bind to readout sequences on primary or encoding nucleic acid probes, and in some cases, the codewords may define an error-correcting code to help reduce or prevent misidentification of the nucleic acid probes.
- a relatively large number of different targets may be identified using a relatively small number of readout probes, e.g., by using various combinatorial approaches.
- Fluorescence microscopy, wide-field fluorescence microscopy, epi-fluorescence microscopy, confocal microscopy, or light-sheet microscopy can be used for image acquisition.
- Image acquisition techniques such as STORM or other super-resolution imaging methods can also be used to image such samples and facilitate determination of the nucleic acid probes. See, e.g., U.S. Pat. Nos. 9,712,805 or 10,073,035, or Int. Pat. Apl. Pub. Nos. WO 2008/091296 or WO 2009/085218, each incorporated herein by reference in its entirety, for additional details regarding techniques such as MERFISH.
- expansion microscopy can also be used in which the sample is expanded before imaging.
- a computer and/or an automated system may be provided that is able to automatically and/or repetitively perform any of the methods described herein.
- automated devices refer to devices that are able to operate without human direction, i.e., an automated device can perform a function during a period of time after any human has finished taking any action to promote the function, e.g. by entering instructions into a computer to start the process.
- automated equipment can perform repetitive functions after this point in time.
- the processing steps may also be recorded onto a machine-readable medium in some cases.
- a computer may be used to control imaging of the sample, e.g., using fluorescence microscopy, wide-field fluorescence microscopy, epi- fluorescence microscopy, confocal microscopy, light-sheet microscopy, diffraction-limited light microscopy, STORM or other super-resolution techniques such as those described herein.
- the computer may also control operations such as drift correction, physical registration, hybridization and cluster alignment in image analysis, cluster decoding (e.g., fluorescent cluster decoding), error detection or correction (e.g., as discussed herein), noise reduction, identification of foreground features from background features (such as noise or debris in images), or the like.
- the computer may be used to control activation and/or excitation and/or deactivation of signaling entities within the sample, and/or the acquisition of images of the signaling entities.
- a sample may be excited using light having various wavelengths and/or intensities, and the sequence of the wavelengths of light used to excite the sample may be correlated, using a computer, to the images acquired of the sample containing the signaling entities.
- the computer may apply light having various wavelengths and/or intensities to a sample to yield different average numbers of signaling entities in each region of interest (e.g., one activated entity per location, two activated entities per location, etc.). In some cases, this information may be used to construct an image and/or determine the locations of the signaling entities, in some cases at high resolutions, as noted above.
- the sample is positioned on a microscope.
- the microscope may contain one or more channels, such as fluidic or microfluidic channels, to direct or control fluid to or from the sample.
- nucleic acid probes such as those discussed herein may be introduced and/or removed from the sample by flowing fluid through one or more channels to or from the sample.
- there may also be one or more chambers or reservoirs for holding fluid, e.g., in fluidic communication with the channel, and/or with the sample.
- channels including fluidic or microfluidic channels, for moving fluid to or from a sample.
- the following examples show a massively multiplexed FISH method for imaging the 3D organization of chromatin at the genome scale in single cells and further demonstrate the ability to place 3D genome organization in its native structural and functional context by combining chromatin and nascent transcript imaging, both at the genome scale, with nuclear- structure identification.
- This first example reports a massively multiplexed FISH approach to allow genome- scale imaging of chromatin organization in single cells. Using this approach, imaging and identification of >1,000 distinct genomic loci (-2,000 chromatin loci counting homologous pairs of chromosomes) across the human genome in single cells was demonstrated.
- RNA transcripts of >1,000 genes residing in these loci were demonstrated in the context of various nuclear structures, including nuclear speckles, nucleoli and nuclear lamina. This approach was used to explore the relationship between chromatin organization, transcriptional activity, and nuclear context in single cells.
- each genomic locus was assigned a unique 100- bit binary code with a Hamming weight of 2, i.e. each barcode containing two “1” bits and 98 “0” bits (Fig. 1A).
- the bit values in these barcodes determined the presence (1) or absence (0) of signal for each locus across sequential rounds of imaging.
- a subset was further selected to encode the targeted genomic loci and optimized assignment of these barcodes, such that loci with a “1” bit in the same barcode position were maximally separated in genomic space. This strategy allowed for minimizing detection errors caused by overlapping signals from nearby chromatin loci.
- this design allowed for identifying and discarding detection errors and further improve measurement accuracy.
- the barcodes were physically imprinted onto the targeted genomic loci using a high- diversity library of encoding probes, each containing a 40-nt target region for binding to one of the targeted loci and a 20-nt readout sequence chosen from 100 pre-designed readout sequences (Fig. 1A).
- Each readout sequence corresponded to one of the 100 bits, and the encoding probe set for each genomic locus (-400 probes per locus) contained only two distinct readout sequences, corresponding to the two bits that read “1” in the barcode assigned to that locus.
- the barcodes imprinted on the chromatin loci were detected by sequential hybridization of fluorescently labeled readout probes, each complementary to one of the 100 readout sequences (Fig.
- 1,041 genomic loci were selected for imaging, each ⁇ 30-kb in size, uniformly covering the 22 autosomes and the X chromosome in human lung fibroblast (IMR90) cells. It was also required that each chromosome contain at least 30 targeted loci, hence the number of loci imaged per chromosome homolog ranged between 30 and 80 depending on the length of the chromosome.
- IMR90 human lung fibroblast
- Fig. IF and Fig. 6A The contact frequencies between pairs of chromatin loci within the same chromosome determined from the imaging data showed high correlation with the contact frequencies detected by ensemble Hi-C, with a Pearson correlation coefficient of 0.91 (Fig. 6B).
- the imaging data captured chromatin structures at multiple scales, from the organization of chromosomes into territories (Fig IF and Fig. 6A) to the formation of A and B compartments within chromosome arms (Fig. 7A), which also agreed well with compartments identified by ensemble Hi-C measurements (Figs. 7B-7C).
- chromosomes By exploring chromatin organization in individual cells, chromosomes, while occupying distinct territories within each cell (Figs. 1F-1G), also displayed substantial overlap with each other (Figs. 1G-1H). On average, -80% of the convex-hull volume occupied by any given chromosome was shared with other chromosomes in the same cell (Fig. II), suggesting a high degree of trans-chromosomal interactions. Since these interactions have been underexplored, the analyses below focused on these trans- chromosomal interactions.
- Figs. 1A-1I show genome-scale chromatin imaging.
- Fig. 1A shows the imaging scheme.
- the targeted genomic loci were assigned error-robust barcodes, e.g. 100-bit binary barcodes with a Hamming weight of 2 (i.e. two of the 100 bits reading “1”).
- the barcodes were imprinted onto the genomic loci with encoding oligonucleotide probes, which recognized the loci and associated two distinct readout sequences with each locus, corresponding to the two bits that read “1” in the barcode assigned to the locus.
- Each locus was labeled by a total of 400 encoding probes, but only 4 are shown.
- Fluorescent readout probes complementary to the readout sequences were sequentially added and imaged, allowing the bits that read “1” at each locus and hence the barcode identity of that locus to be determined. -1000 genomic loci were imaged.
- Fig. IB shows representative images from multiple imaging rounds in the nucleus of a single cell. Fluorescent signal of the chromatin loci from readout probes is shown in a lighter shade, while the signal of 4’,6-diamidino-2- phenylindole (DAPI), used as a nuclear marker, is shown in a darker shade. Scale bar: 5 micrometers.
- Fig. 1C is zoomed-in images of a small spatial region (box in Fig.
- Fig. ID is a 3D rendering of all detected chromatin loci in a single cell, grayscaled according to the chromosomes they belong to. Adjacent loci in genomic sequence are connected by a thin line.
- Fig. IE shows chromatin loci of the same cell as in Fig. ID, but with two homologs of the indicated chromosomes shown in a different greyscale than all other loci.
- Fig. IF is a median distance matrix computed from -5,400 single cells.
- Fig. 1G show example images showing the positions of multiple chromosomes territories in single cells. Shaded areas represent the convex hull surrounding each chromosome, which was used as an operational definition of the chromosome territory.
- Fig. 1H shows distance matrices for the same cells shown in Fig. 1G. The spatial distance between each pair of chromatin loci is shown. Chromosome order is as noted beneath the heatmaps, with the two homologs of each chromosome separately shown.
- Fig. II is a quantification of the fraction of the volume of each chromosome territory that is shared by at least one other chromosome in the same cell.
- n 10,910 copies of chromosomes (5,455 cells and two homologous copies per cell for each chromosome).
- Figs. 6A-6B show contact frequency matrices derived from genome-scale imaging and comparison with ensemble Hi-C data.
- Fig. 6A shows a contact frequency matrix for all 1041 genomic loci imaged in this example. The contact frequency between a pair of loci was calculated as the number of incidences in which the measured distance between the loci is shorter than 500 nm divided by the total number of measured distances between the two loci.
- Fig. 6B shows a correlation plot for the contact frequencies between pairs of loci within chromosomes derived from the imaging data and those derived from ensemble Hi-C experiments, binned at 500 kb and centered around the target loci. The Pearson correlation coefficient was 0.91.
- Figs. 7A-7C show sub-chromosomal structures derived from genome-scale imaging and comparison with ensemble Hi-C data.
- Fig. 7 A shows a contact frequency matrix generated from the imaging data for one arm of chromosome 22. Assignment of each locus to the A or B compartment based on this matrix is shown in the bar beneath the matrix.
- Fig. 7B shows a contact frequency matrix of the same arm of chromosome 22, computed from Hi- C data, binned at 500 kb and centered around the target loci. The bar beneath the matrix shows the A- and B-compartment assignment of each locus based on this matrix, assigned using the same procedure as Fig. 7A. The A/B compartment assignment derived from the imaging data and Hi-C data are identical.
- Fig. 7C shows a correlation plot for the contact frequencies between locus pairs in chromosome 22 derived from the imaging data and those derived from ensemble Hi-C experiments. The Pearson correlation coefficient was 0.91.
- Fig. 8 shows reproducibility of the chromatin imaging experiments between replicates. Shown in the plot is the correlation of pairwise distances between chromatin loci observed in two independent biological replicates of 1041 genomic loci imaging experiments. The Pearson correlation coefficient between replicates was 0.98.
- the upper right cloud represents the trans-chromosomal pairwise distances and the lower-left cloud represents the intra-chromosomal pairwise distances.
- compartment-B loci did not show similar trans-chromosomal affinity towards each other, but instead showed a slightly higher probability to interact with compartment-A chromatin trans- chromosomally (Figs. 2A-2B).
- trans-chromosomal A-A interactions appeared with a substantially stronger tendency than A-B interactions, which in turn appeared with a slightly stronger tendency than B-B interactions.
- compartment-A and compartment-B loci adopted different spatial distributions, with A loci exhibiting a tendency to be more centrally localized than B loci in the nucleus (Fig. 2C and Figs. 9A-9B). There was also a substantial degree of intermixing between A and B loci (Fig. 2C and Figs. 9A-9B).
- Fig. 2C and Figs. 9A-9B For each imaged locus in each chromosome, its local densities of A loci and B loci from all other chromosomes were calculated, and the ratio of these two densities was determined (referred to hereafter as the trans-chromosomal A/B density ratio) (Fig. 2C).
- This quantity provided a measure of the local enrichment of trans-chromosomal active chromatin near the locus.
- the majority (62%) of the imaged loci belonged to compartment-B, creating an overall bias for the A/B ratio to be smaller than 1.
- distributions of the trans-chromosomal A/B density ratios observed for A loci and for B loci were compared with the distribution obtained in a randomization control where the A and B identities of imaged loci were randomly shuffled among the imaged loci, while keeping the numbers of A and B loci unchanged.
- the trans-chromosomal A/B density ratios observed for A loci were substantially higher than the values observed for B loci, which were in turn higher than the values derived from the randomization control (Fig. 2D), and this trend was observed in most single cells (Fig. 2E).
- Figs. 2A-2E show that trans-chromosomal contacts are preferentially enriched for interactions between active chromatin.
- Fig. 2A shows normalized trans-chromosomal contact frequency matrix. The contact frequency between each trans-chromosomal locus pair (pair of loci on different chromosomes) is shown. The loci are reordered such that compartment-A loci appear first, followed by compartment-B loci, hence the top left block represents interactions between pairs of A loci and the bottom right represents interactions between pairs of B loci. Each entry in the matrix is normalized by the median contact frequency of all locus pairs originating from the same pair of chromosomes to account for varying basal levels of interaction between pairs of chromosomes.
- Fig. 1 shows normalized trans-chromosomal contact frequency matrix. The contact frequency between each trans-chromosomal locus pair (pair of loci on different chromosomes) is shown. The loci are reordered such that compartment-A loci appear first, followed
- Fig. 2C shows distributions of compartment-A and compartment-B loci in single cells.
- the left panels represent the locations of all detected loci within a single z-plane in a single nucleus. Compartment-A loci are shown at the top of the scale, while compartment-B loci are shown at the bottom.
- the shade of each locus represents the ratio of the local densities of trans-chromosomal A and B loci, in accordance with the shade scale bar shown on the right.
- Figs. 9A-9B show that compartment-A and compartment-B loci display distinct spatial distributions in single cells.
- Fig. 9A left panels show example images displaying compartment-A loci and compartment-B loci in a single z-plane of single cells.
- the right panel shows the distribution of distances to the nuclear periphery for compartment-A loci and compartment-B loci in these single cells.
- the nuclear periphery is identified as a convex hull surrounding all detected chromatin loci.
- the histogram shows the distribution of distances from the nuclear periphery for points sampled uniformly within the convex hull surrounding the detected chromatin loci.
- the imaging method was extended in this example to allow simultaneous measurements of the chromatin organization together with transcriptional activities of numerous genomic loci as well as landmark structures within the nucleus.
- the 1,041 genomic loci were imaged together with the nascent RNA transcribed from each of the 1,137 genes located at these loci and simultaneously with important nuclear structures, including nuclear speckles and nucleoli (Fig. 3A).
- RNA and nuclear- structure imaging within the same cells multiplexed imaging of the intronic RNAs of the 1,137 genes was performed by adopting a similar combinatorial imaging strategy to the one described above for chromatin (Fig. 3A).
- the RNAs were encoded with a 54-bit, Hamming weight 2 code, and selected 1,137 of the possible barcodes to encode the genes, in a way similar to how the barcodes for chromatin imaging were selected to minimize the chance of imaging spatially proximal genes in the same bit.
- RNA transcripts were enzymatically digested (a step also carried out in our single-modal chromatin imaging experiments) and multiplexed DNA FISH was performed as described above to image the 1,041 genomic loci (Fig. 3 A).
- RNA transcripts were decoding largely independently, with the additional constraint for the transcripts to colocalize with their harboring genomic loci. This procedure further improved detection accuracy for transcribing RNAs and allowed for estimation of the detection efficiency (-90%) for the transcription bursts at each genomic locus.
- nuclear speckles and nucleoli were imaged using immunofluorescence against known molecular components of these structures (Fig. 3A). The positions of nuclear lamina were estimated by computing a convex hull encompassing all imaged genomic loci and determining the boundary of the convex hull. Together, these multi-modal measurements allowed an integrated single-cell view of 3D genome structure, transcriptional activity and nuclear organization (Fig. 3B). These multi-modal imaging experiments were performed on -3700 individual cells, in two biological replicates. Chromatin imaging data from these multi-modal experiments were also included in the 5 replicates (-5,400 cells) described above for 3D genome organization analyses.
- Figs. 3A-3H show genome-scale imaging of chromatin and transcription activity in the context of nuclear structures.
- Fig. 3A is an illustration of the multi-modal imaging scheme that combines chromatin (left panel), nascent RNA transcripts (middle panel) and nuclear bodies (right panel) imaging to generate an integrated view of chromatin organization in the context of nuclear structures and functional activity.
- -1000 genomic loci, nascent RNA transcripts of -1100 genes in the targeted loci, and two types of nuclear bodies (nuclear speckles and nucleoli) are imaged.
- chromatin loci across multiple imaging rounds (left), nascent RNA transcripts across multiple imaging rounds (middle) and nuclear bodies (right: nuclear speckles; left: nucleoli). Scale bar: 5 micrometers.
- Fig. 3B is 3D renderings of chromatin loci, transcriptional bursts and nuclear bodies in a single cell.
- Left All detected chromatin loci, grayscaled by chromosome (based on the chromosome index shown below).
- Middle All detected intronic RNAs shown as spheres, with shading indicating the identities of the imaged genes and sphere size representing transcription burst size. Chromatin loci are shown in the background.
- Right Volume-filling representations of detected nuclear bodies.
- Figs. 3E-3F are scatter plots of the local trans-chromosomal A/B density ratio for each imaged genomic locus as a function of the frequency with which the locus is found associated with the nuclear lamina (Fig. 3E) and nuclear speckles (Fig. 3F).
- a locus is considered associated with a nuclear structure if its measured distance to the structure is smaller than 250 nm.
- the values of trans-chromosomal A/B density ratio shown in the plots are the median values across all imaged cells.
- Fig. 3G shows association frequency with nucleoli for all imaged genomic loci, ordered by genomic position. Vertical lines are the locations of centromeres and brackets highlight chromosomes containing ribosome-encoding genes (rDNAs).
- Fig. 3H shows the effect of nuclear- structure association on transcription.
- Circles are the fold-change in the transcriptional burst frequency for each locus when comparing the populations of cells in which the locus is lamina-associated versus non- lamina-associated (left) and speckle- associated versus non-speckle associated (right).
- the dotted line highlights no change and the solid lines represent the median fold-change in each case.
- Figs. 10A-10B show reproducibility of the nascent RNA transcript imaging experiments between replicates.
- Figs. 10A-10B show the correlation between replicates of RNA imaging for each gene’s burst frequency (Fig. 10A) and burst size (Fig. 10B). Pearson correlation coefficients were 0.94 and 0.81, respectively.
- Fig. 11 shows the preferential association of compartment-B loci with nuclear lamina.
- a locus is operationally defined as being lamina-associated if its distance to the nuclear periphery is smaller than 250 nm.
- Figs. 13A-13C show changes in nuclear lamina and nuclear speckle association upon transcription inhibition.
- Figs. 13A-13B show representative images of individual nuclei with imaged chromatin loci, nucleoli, and nuclear speckles shown for untreated cells (Fig. 13A) and cells treated with the transcriptional inhibitor alpha- amanitin (Fig. 13B). Fig.
- 13C shows the fold change in the rate of associate of each locus with lamina (left) and nuclear speckles (right) upon transcription inhibition by alpha- amanitin.
- the data point for each genomic locus is shown in circles, the solid lines are the median fold changes of all loci in each case, and the dotted line represents no change.
- these multi-modal single-cell measurements were used to further characterize trans-chromosomal interactions in the context of transcriptional activity and nuclear structures. Given the observation that trans-chromosomal interactions were preferentially enriched for interactions between compartment-A loci, it was tested whether these interactions correlate with the transcriptional activity of chromatin. To this end, the local densities of A and B chromatin from other chromosomes and the trans-chromosomal A/B density ratio were calculated for each locus in each cell, and the median values of these quantities for two populations of cells were determined: (i) the cells where the locus under consideration exhibited transcriptional activity (i.e. RNA burst signal), and (ii) the cells where the locus appeared transcriptionally silent at least momentarily (Fig.
- Figs. 4A-4F show preferential trans-chromosomal interactions between active chromatin are correlated with transcription and are disrupted upon treatment that perturbs condensate formation.
- Fig. 4A is single-cell images of chromatin loci and transcriptional activities. Left: Locations of all imaged compartment-A (upper portion of scale) and -B (lower) loci in a single z-plane from a single nucleus. Middle: Local trans-chromosomal A/B density ratios for the same loci, based on the greyscale scale bar. Right: Same as the middle panel, with detected transcriptional bursts overlaid and displayed as circles. Fig.
- FIG. 4B shows a comparison of local trans-chromosomal A/B density ratio for each locus in the transcribed versus silent state.
- the trans- chromosomal A/B density ratio was calculated for the cells in which it was actively transcribed (designated as transcribed) and for the cells in which it was not transcribed (designated as silent). Median values across cells are shown for each state. Loci were ordered by their A/B density ratio in the silent state and the A/B density ratios were plotted for both the silent and transcribed states.
- Fig. 4C shows normalized trans-chromosomal contact frequency matrix for cells treated with alpha- amanitin to inhibit transcription.
- Fig. 4D shows a distribution of AA, BB and AB contact frequencies shown as box plots, as described in Fig. 2B.
- Figs. 4E-4F are the same as Figs. 4C-4D but for cells treated with 1,6-hexanediol.
- Fig. 14 shows the local density of trans-chromosomal A loci near each imaged locus when the locus is in the active transcribed state or the silent state.
- cells were divided into two groups, depending on whether the locus is actively transcribed or silent.
- the median local density of A loci is shown for these two groups of cells (transcribed and silent). The loci are ordered based on their local trans-chromosomal A-locus density in the silent state.
- Figs. 15A-15B show enrichment of active-active trans-chromosomal interactions among chromatin loci not associated with nuclear speckles.
- Fig. 15B shows the fold-change of local trans- chromosomal A/B density ratios between transcribed and silent states for loci not associated with nuclear speckles.
- the fold change in the local trans- chromosomal A/B density ratio between transcribed and silent states of the locus was computed, considering only the cells in which the locus was not associated with a nuclear speckle.
- the median A/B density ratio in each state (transcribed or silent) was determined for each locus and the fold change between the two states is shown on the left (each circle corresponding to a genomic locus).
- the corresponding fold changes derived from all data regardless of nuclear- speckle association status of the loci are shown on the right for comparison.
- the dotted line represents no change and the solid lines represent the median fold change across all loci in each case.
- Figs. 16A-16B show the enrichment of active-active trans-chromosomal interactions among chromatin loci associated with nuclear lamina.
- Fig. 16B shows the fold-change of local trans-chromosomal A/B density ratios between transcribed and silent states for loci associated with nuclear lamina. For each genomic locus, the fold change in the local trans-chromosomal A/B density ratio between transcribed and silent states of the locus was computed, considering only cells in which the locus was associated with the nuclear lamina.
- the median A/B density ratio in each state was determined for each locus and the fold change between the two states is shown on the left (each circle corresponding to a locus). Outliers (33 loci above and 18 loci below the presented scale) were omitted to allow a clearer visualization of the median fold change.
- the fold changes derived from all data regardless of lamina-association status are shown on the right for comparison.
- the dotted line represents no fold change and the solid lines represent the median fold change across all loci in each case.
- RNA polymerase II contains low-complexity domains (LCD) and can form condensates
- alpha-amanitin a transcription inhibition drug
- these examples developed a massively multiplexed FISH method for imaging the 3D organization of chromatin at the genome scale in single cells and further demonstrated the ability to place 3D genome organization in its native structural and functional context by combining chromatin and nascent transcript imaging, both at the genome scale, with nuclear-structure identification.
- This provides an integrated view of nuclear organization in single cells.
- target loci were chosen uniformly across all chromosomes here to provide an unbiased view of the overall 3D genome organization, this method could also be used to target genomic loci with specific structural and functional properties, such as promotors, enhancers, and loci bound by specific nuclear architecture proteins, to study the interactions among these loci and their relationship with transcription and other chromatin functions.
- the broad applications of this approach to a wide range of questions related to genome organization could illuminate both the mechanisms governing chromatin organization and the role of chromatin structures in regulating genome functions.
- This example illustrates various materials and methods usable in the above examples.
- Target genomic regions For chromatin imaging, genomic loci were chosen for imaging in the following way. For each human autosome and X chromosome, a 30-kb segment every ⁇ 3 Mb of spacing was selected. If this spacing resulted in less than 30 selected loci on a given chromosome, the spacing was reduced for that chromosome, until all chromosomes had at least 30 loci selected. This resulted in a total of 1,041 target genomic loci for imaging, and the number of loci in individual chromosomes ranged from 30-80. Encoding probes were then designed for each 30-kb segment (-400 oligonucleotide probes) for the combinatorial FISH imaging.
- RNA transcripts For imaging of nascent RNA transcripts, all intron-containing genes that completely or partially overlap with the targeted genomic loci were selected. Encoding probes were then designed for the introns of all of these RNAs such that each RNA had -20 encoding probes and that the targeting sequences of the encoding probes were kept as close as possible to the transcription start site (TSS). A total of 1,137 genes were targeted.
- Barcode design for combinatorial FISH imaging Binary barcodes for imaging the 1,041 genomic loci were chosen in the following fashion. First, all possible 100-bit binary barcodes with a Hamming weight of 2 (i.e. each barcode containing two “1” bits and 98 “0” bits) were generated and 1,041 barcodes from this list were randomly selected. The selected barcodes were then arbitrarily assigned to the 1041 genomic loci first. Next, barcodes were exchanged randomly between the used and unused code pool, as well as between loci from different chromosomes, in order to minimize, for each chromosome, the variance in the number of loci appearing (i.e. reading “1”) across different bits.
- loci within the same chromosome were allowed to exchange barcodes and optimized for the largest minimal genomic distance between loci with barcodes reading “1” at the same code position.
- code assignments with identical minimal genomic distances the one that minimized the coefficient of variation of genomic distances was selected (so that genomic distances have both larger means and smaller standard deviations).
- Barcodes for imaging the nascent RNA transcripts of the 1,137 genes were chosen similarly, but using a 54-bit, Hamming distance 2 code instead of a 100-bit, Hamming distance 2 code.
- Encoding probe design Encoding probes for chromatin imaging were synthesized from a pool of oligonucleotides purchased from Twist Biosciences. Each oligo in this pool used the following sub-sequences (from 5’ to 3’):
- a 40-nt target sequence designed to bind uniquely to a single targeted genomic locus
- the forward and reverse priming sequences were chosen from a previously generated list of random 20-nt sequences optimized for PCR.
- the readout sequences were chosen via the following process. First, a list of 30-nt sequences with minimal homology to the human genome was created. Then, a subset of these sequences were ranked by observed signal to noise ratio (SNR) and the top 100 were chosen as DNA readout probes. Lastly, the readout sequences were chosen by reverse complementing the last 20 nt of each of these sequences.
- SNR signal to noise ratio
- the 40-nt target sequence was chosen similarly. Briefly, the following procedure was repeated for each genomic region of interest (see the “Target genomic regions” discussion above). First, a list of all 40-nt sequences complementary to the genomic region of interest was created (starting at each possible base in the targeted region). Then, sequences were filtered by requiring them to be within a defined range of melting temperatures and GC content. The remaining sequences were then further filtered by limiting the allowed degree of homology to the human genome, the human transcriptome and a database containing repetitive sequences. Homology was determined by creating a table of all possible 17-nt sequences and the number of times they appear in the target database (e.g.
- target sequences were selected from the remaining sequences after the final filtering step such that no genomic overlap exists between any pair of target sequences.
- each of the chosen 40-nt target sequences for each target genomic locus was alternatingly assigned to 2 groups spanning the entire target locus. Each of these groups were associated with a single readout sequence, corresponding to one of the two bits in which the locus would be imaged. Then, each target sequence was concatenated to two identical copies of the readout sequence assigned to its group, and then concatenated to the forward and reverse PCR primers.
- Probes for RNA imaging were designed similarly, with the exception that they contained 3 copies of an identical readout sequence on every probe, one at the 5’ end and two at the 3’ end of the target region. Readout sequences for RNA imaging were orthogonal to those used for DNA imaging and were selected from the same ranked list of tested readout sequences.
- Encoding probe synthesis Encoding probes were amplified from the template library described above (see “Encoding probe design” above). This was done using an amplification protocol involving the following steps:
- the initial oligo pool was expanded using limited-cycle PCR for approximately 20 cycles.
- the reverse primer used in this step also introduced a T7 promoter sequence via primer extension.
- the resulting product was purified via column purification and underwent further amplification and conversion to RNA by a high-yield in-vitro transcription reaction.
- RNA product was converted back to single-stranded DNA by a reverse transcription reaction.
- the product of the previous step was dried in vacuum and resuspended in water to achieve the desired concentration of primary probe.
- IMR-90 cells were purchased from American Type Culture Collection (ATCC, CCL-186) and grown according to the recommended protocol. To avoid potential alterations to chromatin structure, all cells in this study were plated within 6 weeks of culture initiation at the density specified below.
- coverslips were coated with 10 micrograms/mL fibronectin (Sigma-Aldrich, FI 141) prior to cell plating and replaced media with fresh media containing 2% w/v 1,6-hexanediol for 45 minutes.
- the culture was then fixed using 4% paraformaldehyde (PFA) in PBS for 10 minutes at room temperature and washed in PBS 2-3 times.
- PFA paraformaldehyde
- Cells were then permeabilized in two steps: first, they were treated with 0.5% v/v Triton-X (Sigma-Aldrich, T8787) in PBS for 10 minutes at room temperature.
- HC1 hydrochloric acid
- PBS 0.1 M hydrochloric acid
- HC1 hydrochloric acid
- cells were treated with a solution of 0.1 mg/mL RNase A (ThermoFisher, EN0531) dissolved in PBS for 30-45 minutes at 37 °C, to remove potential sources of off-target binding to RNA.
- RNase A ThermoFisher, EN0531
- cells were incubated in pre -hybridization buffer, consisting of 2x saline-sodium citrate buffer (SSC; Ambion, AM9763) and 50% formamide (Ambion, AM9342) for approximately 10 minutes.
- SSC 2x saline-sodium citrate buffer
- AM9342 50% formamide
- the cell coverslip was inverted and placed on a drop of 50 microliters of hybridization buffer (2x SSC, 50% formamide, 10% dextran sulfate (Sigma-Aldrich, D8906) containing a mixture of encoding probes at -25 micmolar total concentration with or without 10 micrograms Human Cot-1 DNA (ThermoFisher, 15279011)) in a 60-mm petri dish.
- the dish was partially submerged in a water bath at -90 °C for 3 minutes and incubated at 47 °C in a humidified chamber for 16-36 hours.
- the sample was washed in 2x SSC and 40% formamide for 30 minutes and post-fixed with 4% PFA in 2x SSC for 10 minutes at room temperature.
- the sample was then incubated for 2-3 minutes with fiducial beads (either ThermoFisher F8805 or ThermoFisher F8792) in 2x SSC and stained with 1 micromolar 4’,6-diamidino-2-phenylindole (DAPI; ThermoFisher D1306) in 2x SSC for 5-10 minutes, and then stored in 2x SSC until imaging.
- fiducial beads either ThermoFisher F8805 or ThermoFisher F8792
- RNA staining was identical to the above-described protocol up to treatment with HC1. After this step, cells were incubated in pre-hybridization buffer for 10 minutes, and the cell coverslip was then inverted and placed on a drop of hybridization buffer containing encoding probes targeting the RNA introns at -1 micromolar total concentration, as described for DNA staining. In this case, however, no 90 °C heat denaturation was performed, and cells were immediately incubated at 47 °C in a humidified chamber for 16-36 hours.
- RNAse inhibitor either NEB M0314 or Fisher Scientific N2615
- the sample was washed in a formamide solution and post-fixed with PFA as described for DNA above. It was then incubated with fiducial beads and stained with 1 micromolar DAPI, before being stored in 2x SSC until imaging. After RNA imaging, the sample was removed from the microscope, the cells were treated with RNase A and then the DNA hybridization proceeded in the same manner as described above for DNA imaging without RNA imaging.
- the imaging buffer was prepared and included 60 mM Tris pH 8.0, 10% w/v glucose, 1% Glucose Oxidase Oxygen Scavenger Solution (containing -100 mg/mL Glucose Oxidase (Sigma-Aldrich, G2133) and a 1:3 dilution of catalase (Sigma-Aldrich, C3155)), 0.5 mg/mL 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox; Sigma-Aldrich, 238813) and 50 micromolar Trolox Quinone (generated by UV irradiation of a Trolox solution). Trolox was dissolved in methanol before being added to the solution. After preparation, the imaging buffer was covered by a -0.5 cm thick layer of mineral oil to prevent exposure to oxygen.
- the hybridization buffer and wash buffer were made up of 35% and 30% formamide in 2x SSC, respectively, with the hybridization buffer also containing 0.01% v/v Triton-X.
- the hybridization buffer was kept separately for each hybridization round and contained two or three (for DNA and RNA imaging, respectively) sets of readout probes. Fluorescent signal was introduced in one of two ways:
- hybridization buffer included two fluorescent readout probes, one labeled with Cy5 or Alexa647 and the other labeled with Alexa750.
- Fluorescent readout probes used either: 1) a fluorescently labeled oligo complementary to a readout sequence common to all encoding probes imaged in a given bit, added at 100 nM concentration, or 2) a combination of an adaptor oligo having the sequence complementary to a readout sequence, concatenated to an additional readout sequence (referred to as the secondary readout sequence) common to all adaptors (more accurately, common to all adaptors in each color channel) and orthogonal to all other used readout sequences, and a fluorescently labeled oligo probe complementary to this secondary readout sequence.
- the adaptor and secondary readout probes were pre-mixed in a 1:1.5 ratio and added to a final concentration of -100 nM.
- the adaptor and readout probes were hybridized sequentially to the sample. This allowed for using lower concentration of the more expensive secondary readout probe.
- each round s hybridization buffer contained three adaptor oligos (to be detected in three different color channels), each binding to a different readout sequence and each containing an additional, secondary readout sequence. All adaptors corresponding to the same color channel shared the same secondary readout sequence.
- Each round included two discrete hybridization steps: first the adaptors were flowed in, hybridized, and excess material was washed. Then three fluorescent readout probes, complementary to the secondary readout sequences on the adaptors, respectively labeled with Cy3, Cy5, and Alexa750, were flowed in sequentially. Fluorescent readout probes used for RNA imaging contained a disulfide bond linking the fluorophore to the secondary oligo, to allowefficient removal of signal between rounds. After the fluorescent readouts were hybridized, imaging buffer was flowed in and signal was collected.
- a round of imaging was performed to acquire the DAPI signal and identify nuclear boundaries. Then, the entire set of 1,041 genomic loci were imaged in 50 rounds of hybridization and 2 color channels per round. In each round, the genomic loci were imaged in 3D by stepping the stage in the z-dimension. Nascent RNA transcripts for 1,137 genes were imaged likewise in 3D in 18 rounds in 3 colors. Additional rounds were used to relabel sets of genomic loci and assess chromatic aberration and bleedthrough between color channels. Imaging of -60 fields of view containing a total of -1,000-2,000 cells took -3 days. The 3-4 valve system allowed loading up to 20-30 different hybridization solutions.
- the sample chamber was bypassed and all the channels used for hybridization were washed with 30% formamide in water. Next, the chamber was reconnected and the next set of hybridization and imaging rounds were performed.
- Antibody labeling and imaging was performed immediately after RNA or DNA imaging. Following completion of imaging via the protocols described above, samples underwent the following steps:
- Steps 2 and 3 were repeated for a fluorescently-tagged secondary antibody
- a primary antibody was used against SC35 (Abeam, abl 1826) - a splicing factor commonly used as a marker of nuclear speckles - at 1:200 dilution from stock and a donkey anti-mouse secondary antibody labeled by a Cy5 dye (Jackson Immunoresearch, 715-175-150) diluted 1:1,000 from stock concentration.
- anti-fibrillarin antibody was used (Abeam, ab5821), at 1:200 dilution from stock, and a donkey anti-rabbit secondary antibody labeled by an Alexa 657 dye (Jackson Immunoresearch, 711-605-152), diluted 1:1,000 from stock concentration.
- the signal from the previous round was extinguished. This was achieved via photobleaching of the signal. Photobleaching was performed by changing the buffer to 2x SSC and illuminating each field of view with the maximum available power of the 647 and 750 lasers (as well as the 560 laser when imaging RNA) for 10 seconds.
- the buffer used for bleaching also contained 50mM tris(2-carboxyethyl)phosphine (TCEP; Sigma-Aldrich, C4706) to cleave the disulfide bond connecting fluorophores to readout probes.
- TCEP tris(2-carboxyethyl)phosphine
- Image acquisition was performed using a custom-built microscope system. The system was built around a Nikon Ti-U microscope body with a Nikon CFI Plan Apo Lambda 60x oil immersion objective with 1.4 NA. Illumination was based on one of two alternatives:
- Solid-state, single-mode lasers with the following wavelengths: 405 nm (Coherent, Obis 405 nm LX 200 mW), 560 nm (MPB Communications, 2RU-VFL-P-2000-560-B1R), 647 nm (MPB Communication, 2RU-VFL-P-1500-647-B1R) and 750 nm (MPB Communication, 2RU-VFL-P-500-750-B1R).
- the output of the 560-nm, 647-nm and 750-nm lasers were controlled by an acousto-optic tunable filter (AOTF) while the 405- nm laser was controlled directly via its laser control box.
- AOTF acousto-optic tunable filter
- a custom dichroic (Chroma, zy405/488/561 /647/752RP-UF 1 ) and emission filter (Chroma, ZET405/488/461/647- 656/752m) were used to separate excitation and emission illuminations.
- a Lumencor CELESTA light engine (a fiber-coupled solid-state laser-based illumination system) with the following wavelengths: 405 nm, 446 nm, 477 nm, 520 nm, 546 nm, 638 nm and 749 nm. This system was used with a penta-bandpass dichroic (IDEX, FF421/491/567/659/776-Di01-25x36) and a penta-bandpass filter (IDEX, FF01- 441/511/593/684/817-25).
- IDEX penta-bandpass dichroic
- IDEX penta-bandpass filter
- CMOS camera Hamamatsu FLASH4.0 or Hamamatsu C 13440 with factory calibration for single-molecule imaging
- Sample position in three dimensions was controlled using a XYZ stage (Ludl).
- a custom-built auto focus system was used to maintain a constant focal plane over prolonged periods of time.
- Each camera FOV had 1,000 x 1,000 pixels, with a camera pixel corresponding to 153 nm in each dimension in the imaging plane, or 2048 x 2048 pixels, with a camera pixel corresponding to 108 nm in each dimension in the imaging plane.
- z-stack images of each FOV were acquired in 3 or 4 colors: 647 nm and 750 nm illumination (or 560 nm, 647 nm, and 750 nm illumination for RNA imaging in the case of combined DNA and RNA imaging) were used to acquire FISH images, 560 nm illumination (or 405 nm illumination in the case of combined RNA and DNA imaging) was used to image fiducial beads.
- 647 nm and 750 nm illumination or 560 nm, 647 nm, and 750 nm illumination for RNA imaging in the case of combined DNA and RNA imaging
- 560 nm illumination or 405 nm illumination in the case of combined RNA and DNA imaging
- 405 nm illumination was used to image the DAPI signal
- the 647 nm excitation channel was used after RNA or DNA imaging.
- Consecutive z-sections were separated by 85, 100 or 150 nm, covering the entirety of the nuclear volume for all imaged cells. At each z position, images were acquired in all channels before the stage was moved and images were acquired at a rate of -10 Hz.
- DAPI signal was used to identify the borders of individual nuclei, as well as for image registration between RNA and DNA imaging
- Fitted spots were compared with other localizations within the same nucleus across all rounds of hybridization to identify the loci from which they originated, using a custom algorithm and software (described in detail in “Decoding algorithm for fitted DNA spots” and “Decoding algorithm for fitted RNA spots” sections).
- Fiducial bead spot fitting was performed in the same way as described above. The set of fiducial bead positions was then compared between rounds of hybridization and a rigid transformation was applied to minimize the sum of square difference of the relative position of beads.
- Bleedthrough and chromatic aberration for multi color imaging were performed by labeling the same set of genomic loci in each imaging channel independently and comparing the signals of the same loci in the different channels, respectively.
- Nuclei segmentation DAPI images from the first round of imaging were used to identify the volume of individual nuclei and allowed for cell segmentation. This was achieved via a convolutional neural network, built and trained, which took the maximum projection of the DAPI image onto the xy plane as input.
- nuclear bodies from immunofluorescence imaging.
- the location of nuclear bodies was extracted from immunofluorescence signals by applying a threshold to the intensity of the immunofluorescence signals, resulting in a pixelized mask identifying high immunofluorescence signals. This was then treated as a pixelized set of locations “containing” nuclear bodies.
- step 5 For each spot pair the three quality metrics in step 3 were combined into a single measure by calculating the combined Fisher p-value for every candidate spot pair against the “valid distributions” in step 4. This was thought of as the overall quality score of each spot-pair, and was calculated per pair in the following way: for each of the three metrics the fraction of other spot-pairs in the “valid distribution” was calculated with lower quality metric and multiplied these fractions together. An expectation-maximization procedure was then used to sequentially select the two spot-pairs with the highest quality score corresponding to each targeted chromatin locus and reupdated the “valid distribution,” and this optimization procedure was repeated until convergence. After convergence, the final sets of spot pairs, each corresponding to a chromatin locus, were used to determine the 3D spatial positions of the loci.
- step 5 a modified K-means algorithm was used to separate the chromatin loci belonging to the same chromosome into two homologs.
- points were switched progressively between the groups to first maximize the fraction of assigned points in each homolog and then the radius of gyration of each homolog was minimized.
- step 7 After separating the two homologs their center of mass and the distance of each spot-pair from step 2 were calculated to their parent chromosome’s center of mass. The distance to the chromosome center was added as another quality metric in addition to the 3 metrics considered in step 3 and steps 3-6 were repeated.
- RNA bursts were compared again to the location of the DNA locus harboring the gene to which they decode, this time with the refined image registration. If the nascent RNA localization was within a cutoff distance from its harboring DNA locus at this stage, it was considered as a detected transcriptional burst.
- the position of the nuclear lamina was estimated by generating the minimal 3D convex hull (using Python’s scipy package) surrounding the locations of all decoded chromatin loci in a given cell.
- Spatial distance The spatial distance between any pair of loci was simply calculated as the Euclidean distance between their fitted 3D Gaussian centers, multiplied by the appropriate ratios relating camera pixels and z steps to physical distance.
- the minimal Euclidean distance to all identified nuclear body “locations” or the minimal distance to the surface of the convex hull defining the nuclear lamina was calculated.
- the density contribution of each other locus was calculated form a different chromosome by evaluating a Gaussian function value with a standard deviation of 500 nm (adjusted to account for variability in cell size) at the distance between the two loci.
- the total A density at the locus was then computed as the sum of this Gaussian function value for all trans-chromosomal A loci, and the total B density was computed in an analogous way.
- the total density of trans-chromosomal compartment-A loci was divided by the density of trans-chromosomal compartment-B loci to find the A/B density ratio at the locus.
- step 2 From the total set of chromatin loci identified in step 1, the fraction (/) of loci that colocalized with RNA signal from exactly one of its gene’s corresponding bits (and not with both bits) were determined. From the measured/(8.4%), which should be equal t ) was estimated.
- Hi-C data analysis Hi-C data for IMR-90 cells was procured and loaded using a straw. For identification of A/B compartments in individual chromosomes, established published protocols were followed. For comparison of contact frequencies derived from the imaging data to Hi-C, bins centered around the targeted regions were created and Hi-C data for these bins was procured by summing the number of reads in higher resolution Hi-C data.
- 3D three-dimensional (3D) organization of chromatin regulates many genome functions.
- the understanding of 3D genome organization is, however, hindered by a lack of tools to directly visualize chromatin conformation in its native context.
- Reported herein is an imaging platform for visualizing chromatin organization across multiple scales in single cells with high genomic throughput.
- multiplexed imaging of hundreds of genomic loci by sequential hybridization was demonstrated, which allowed high-resolution conformation tracing of whole chromosomes.
- a combinatorial imaging method for genome-scale chromatin tracing was developed and demonstrated simultaneous imaging of >1000 genomic loci and nascent transcripts of >1000 genes together with landmark nuclear structures.
- chromatin is partitioned into genomic regions with enhanced self-interaction, termed topologically associated domains (TADs), which appear as block-like structures on Hi-C contact maps.
- TADs topologically associated domains
- TADs ranging from hundreds of kilobases (kb) to several megabases (Mb) in size
- chromatin is partitioned into two major compartments, called A and B compartments, which are respectively enriched for active and inactive chromatin, as revealed by an alternating “plaid” pattern in Hi-C maps, consistent with previous imaging-based observations that gene- rich and gene-poor segments of chromatin tend to spatially segregate.
- Recent imaging experiments show that compartment A and compartment B chromatin indeed tend to spatially segregate in single cells.
- the physiological significance of A/B compartmentalization is implicated by its changes during development and between cell types.
- Imaging-based approaches provide a direct measure of the spatial positions of chromatin loci in individual cells with a high detection efficiency.
- fluorescence in-situ hybridization allows highly specific detection of chromatin loci in fixed cells and, more recently, the clustered regularly interspersed short palindromic repeats (CRISPR) system substantially enhanced the ability to image specific chromatin loci in live cells.
- Chromatin imaging can also be combined with RNA and protein imaging to investigate the interplay between chromatin organization and transcriptional activity or interacting protein factors.
- current imaging methods have limited throughput in genomic (sequence) space, traditionally allowing the study of only a few different genomic loci at a time.
- the sequential imaging approach was substantially advanced to allow imaging of hundreds of genomic loci and this method was applied to provide a high-resolution view of entire chromosomes, elucidating chromatin domain and compartment structures, their relationship with each other, as well as the relationship between chromatin organization and transcription in single cells.
- a massively multiplexed FISH approach was developed based on combinatorial labeling and imaging, which allowed more genomic loci to be imaged with much fewer hybridization rounds.
- an imaging platform using a custom microscope and fluidics setup (see Example 19), was developed for direct visualization of chromatin with exceptionally high throughput in sequence space, up to the genome scale.
- This platform included two complementary approaches (Fig. 17A).
- Fig. 17A First, for imaging of chromatin structures that were relatively small, such that different loci contained therein would be difficult to resolve in any single image, the previously reported sequential imaging strategy was expanded to allow tracing of hundreds of chromatin loci in single cells. In this approach, chromatin was imaged one locus at a time (or 2-3 loci at a time with 2-3 color imaging) across many imaging rounds (Fig. 17A, left).
- FIG. 17A-17M show high-resolution whole-chromosome tracing by sequential hybridization and characterization of chromatin domains in single cells.
- Fig. 17A shows schematics of the multi-scale chromatin tracing platform.
- Left Schematic of chromatin tracing of whole chromosomes by sequential hybridization and imaging. When the target chromatin structure is comparable to or smaller than the diffraction limited resolution, a single chromatin locus is imaged in each color channel per imaging round. After all rounds of imaging, a chromatin trace can be generated in 3D for each copy of the targeted chromosome.
- Right Schematic of genome-scale imaging by combinatorial FISH.
- target loci When target loci are expected to be spread out in a space that is substantially larger than the diffraction limited resolution, such as when loci are dispersed in the entire nucleus, multiple loci can be imaged and resolved in each round, and the identity of each locus can be derived from a barcode based on the combination of imaging rounds in which the locus is detected. This approach significantly reduces the number of rounds required to image the same number of loci compared to the sequential imaging approach.
- chromosome 21 Human chromosome 21 (Chr21) was focused on first and the non- repetitive portion of the chromosome (Chr21: 10.4 - 46.7 Mb) was partitioned into >600 contiguous segments (i.e. >600 genomic loci), each 50-kb in length.
- a library of primary oligonucleotide probes was designed, each containing a variable target sequence for hybridizing to the chromosome and a readout sequence that was unique to each of the 50-kb loci (Fig. 24 A).
- a two-step labeling strategy was devised to detect the distinct readout sequences with a common set of three dye-labeled oligonucleotide probes (called readout probes, one readout probe for each color channel), mediated by unlabeled adaptor probes that convert each locus-specific readout sequence into one of the three common readout sequences (Fig. 24A).
- readout probes one readout probe for each color channel
- unlabeled adaptor probes that convert each locus-specific readout sequence into one of the three common readout sequences
- the imaging protocol was further optimized in the following ways: (i) to maintain sample integrity and primary probe binding stability, the sample was re-fixed with formaldehyde periodically during the course of imaging; (ii) to ensure complete removal of fluorescence signal after each imaging round and minimize the accumulation of residual signal across hundreds of labeling rounds, a combined chemical cleavage and photobleaching approach was used to remove the fluorescence signal of the readout probes, and unlabeled readout probes were added to block any unoccupied binding sites on adaptor probes after each round of imaging; (iii) to minimize perturbations and the experiment time, the duration of each hybridization round and the flow rates of the fluidics system were optimized.
- each chromatin locus was determined in 3D and the conformation of each homologous copy of Chr21 in each cell was reconstructed (Fig. 17B).
- the same chromatin loci were reimaged after different numbers of hybridization/imaging rounds and the displacement between the original loci’s locations and those of the corresponding re-imaged loci were used as a measure of the measurement accuracy.
- the median displacement between the original and re-imaged loci increased from -70 nm when 11 hybridization rounds separated the two imaging instances to -120 nm when the initial and re-imaging instances were separated by -250 hybridization rounds (Fig.
- Fig. 17B shows 3D structural rendering and spatial distance matrices of the two copies of Chr21 in a single IMR90 cell imaged by the sequential hybridization approach.
- Fig. 17C shows ensemble proximity frequency matrix of Chr21 and preferential positioning of single-cell domain boundaries at CTCF/RAD21 -binding sites.
- Top Proximity frequency matrix for Chr21 derived from imaging data. Each matrix element is defined as the frequency with which the measured distance between a pair of loci is shorter than a cutoff distance of 500 nm.
- Middle zoomed-in version of the proximity frequency matrix for a 10- Mb portion of Chr21.
- Bottom the probability of single-cell domain boundary formation at each of the imaged 50-kb genomic segment.
- Triangles show CTCF and RAD21 ChIP-seq peaks.
- Figs. 24A-24N show high-resolution whole-chromosome tracing by sequential hybridization, and ensemble statistics of Chr21 structural features in comparison with Hi-C.
- Fig. 24A shows labeling and imaging scheme for sequential hybridization with adaptor probes.
- the sample is hybridized with primary probes, each containing a target sequence that allows specific binding to a targeted genomic locus and a readout sequence.
- Each locus is labeled by a total of 350-500 primary probes, but only one is shown.
- Each targeted genomic locus is assigned a unique readout sequence (shown in various colors), common to all primary probes that bind to the locus.
- the readout sequences are then detected using sequential rounds of hybridization.
- the readout sequences corresponding to the targeted loci are labeled with oligonucleotide adaptor probes that each consist of two portions: a segment complementary to the locus -specific readout sequence and a segment containing a color-channel- specific common readout sequence.
- Each color channel contains a unique common readout sequence that is shared by all the adaptors visualized in the same color channel.
- the common readout sequences are then hybridized to dye-conjugated, complementary readout probes in corresponding color channels. This procedure allows three genomic loci to be imaged in three color channels during each round of hybridization. Following each round of imaging, fluorescent dyes, which are connected to the readout probes by a di- sulfide bond, are cleaved from the common readout probes by TCEP, and any unoccupied readout sequences on the adaptors are blocked with unlabeled common readout probes to prevent crosstalk between rounds of hybridization. The process is iterated over hundreds of rounds until the detection of all readout sequences, and hence all targeted genomic loci, is completed.
- Fig. 24B shows the displacement of loci over the course of a single experiment. Consecutive 50-kb segments within a 900-kb region in Chr21 (chr21:32.45-33.35Mb) were imaged both at the beginning and at the end of the experiment, separated by > 250 rounds of hybridization. The distribution of the displacement between the re-imaged spot and its originally imaged counterpart are shown. For comparison, the distribution of the distances between adjacent 50-kb segments in the same 900-kb region measured in the original imaging rounds are shown.
- Fig. 24C shows boxplots of the displacement of chromatin loci between the original and re-imaging rounds separated by different number of hybridization rounds. The medians (center lines), 25th - 75th percentiles (boxes) and 10th - 90th percentiles (whiskers) are shown.
- Fig. 24D shows boxplots of the fluorescent signal of chromatin loci with low ( ⁇ 500 nm) and high (>500 nm) displacements errors between original and re-imaging experiments separated by >250 rounds of hybridization.
- the medians (center lines), 25th - 75th percentiles (boxes) and 10th - 90th percentiles (whiskers) are shown.
- Fig. 24F shows a comparison of median spatial distance matrix derived from imaging (left), proximity frequency matrix derived from imaging (middle), and ensemble Hi-C contact matrix (right) for Chr21.
- two chromatin loci are considered to be in proximity when the spatial distance between the two loci is smaller than a cutoff distance of 500 nm.
- the Hi-C contact matrix is binned at 50 kb and centered around the target regions.
- Fig. 24H is the same as Fig. 24F but for a 3-Mb region in Chr21 (chr21: 30.30-33.38 Mb). TAD boundaries are marked with lines.
- Fig. 24J shows a Pearson correlation between Hi-C contact map and imaging-derived proximity frequency maps generated with varying cutoff distances.
- a cutoff distance is chosen and two loci with a distance smaller this cutoff value are considered to be in proximity. Then, proximity frequency between a pair of loci was calculated as the number of incidences in which the measured distance between the loci is shorter than the cutoff distance divided by the total number of measured distances between the two loci.
- Fig. 24K shows normalized insulation scores as a function of the genomic position on Chr21 calculated from: 1) the median pairwise distances from imaging (top ), 2) proximity frequencies from imaging (middle ), and 3) Hi-C contact reads (bottom).
- a fixed-length (250 kb) genomic segment upstream and a same-length segment downstream of the position of interest were first selected.
- the normalized insulation score is then defined as the difference between median inter-segment pairwise distance and median intra-segment pairwise distance, normalized by the sum of these two median distances.
- TAD boundaries are defined as local maxima of the normalized insulation score along the chromosome, identified by a standard peak-calling algorithm (see Example 19).
- the vertical dotted lines are the ensemble TAD boundaries called from the Hi-C data. Also shown in the top and middle panels are the median distances (black line, top panel) and proximity frequencies (black line, middle panel) after perturbing the loci positions with a 3D Gaussian noise term with a standard deviation of 100 nm, comparable to the estimated localization measurement error.
- Fig. 24L shows chromatin domains in a 10-Mb region of Chr21 (chr21: 28.2-38.1Mb) in two example single cells. Pairwise distances from two individual copies of Chr21 in single cells are shown (Top, middle) together with population median pairwise distances derived from all image cells (bottom).
- Chromatin domains in single chromosomes were partitioned into domains that manifest as block-like features in single-cell spatial distance matrices (Fig. 24L). These domains and the inter- loci distances showed high variability from cell to cell (Figs. 24L-24M), consistent with the substantial cell- to-cell variability in chromatin contacts observed in single-cell Hi-C data. Similar domain structures in single cells were previously observed when imaging small ( ⁇ 2 Mb) regions of the chromosome with similar resolution.
- genomic locations of the boundaries of these single-cell domains were first identified and quantified the probability of boundary formation at each 50-kb genomic locus. While a non-zero probability of boundary formation was observed at all imaged genomic loci, the domain boundaries were preferentially positioned near the binding sites of CTCF and cohesin (Figs. 17C-17D).
- Fig. 17D average probability of domain boundary formation in single cells at genomic locations centered around CTCF/Rad21 -binding sites or around ensemble TAD boundaries (grey).
- Fig. 17E is an example of two single-cell chromatin domains with identical genomic coordinates, occupying large (top) or small (bottom) volumes in the physical space.
- Left 3D rendering of the chromatin domains, with green balls representing imaged genomic loci within the domain and flexible linkers connecting adjacent loci in genomic sequence. Grey spots represent imaged loci in the rest of the chromosome. Scale bar: 1 micrometer.
- Right pairwise distance matrix for the chromatin domain shown on the left (marked with lines), with flanking regions.
- Fig. 17F shows an example of two pairs of chromatin domains with high (top) and low (bottom) insulation scores.
- Scale bar 250 nm.
- Fig. 17G shows two examples of long-range contact between chromatin domains with partially overlapping volumes.
- Left 3D rendering of the chromatin domains, as in (E). Shadings represent different domains.
- Scale bar 250 nm.
- the grey space indicates a gap in genomic distance of 22.85 Mb.
- Fig. 17H shows an example of chromatin domains flanked by CTCF binding sites, showing small (top) and large (bottom) distances between the CTCF sites.
- Left 3D rendering of the chromatin domain, as in (E), but with the loci of CTCF sites at the domain ends.
- Scale bar 250 nm.
- Right pairwise distance matrix for the chromatin domain with domain and border CTCF marked correspondingly.
- Fig. 171 shows a distribution of measured genomic sizes of chromatin domains in Chr21 in single cells. Shown in black line is the distribution of genomic sizes of chromatin domains in Chr21 in single cells derived from simulated data that considers a localization error of 100 nm. In this simulation, the positions of the imaged loci are perturbed with a 3D Gaussian noise with standard deviation of 100 nm, similar to our measurement error.
- Fig. 17J shows a distribution of measured physical sizes, as defined by the radii of gyration, of chromatin domains in Chr21 in single cells. Shown in black line is the distribution of physical sizes of chromatin domains in Chr21 in single cells derived from simulated data that considers a localization error of 100 nm, as in Fig. 171.
- Fig. 17K shows a median radius of gyration as a function of genomic size for chromatin domains with boundary loci containing interacting CTCF/Rad21 sites and with neither boundary locus containing CTCF/Rad21 sites. Error bars indicate 95% confidence intervals derived by resampling.
- Fig. 17L shows a distribution of insulation scores between neighboring domains with domain boundaries occurring at CTCF/Rad21 -binding sites and non-CTCF/Rad21 -binding sites.
- Fig. 17M shows a median of normalized end-to-end distance of domains as a function of genomic size for chromatin domains with boundary loci containing interacting CTCF/Rad21 sites and with neither boundary locus containing CTCF/Rad21 sites.
- the normalized end-to-end distance is defined as the domain’s end-to-end distance divided by the median distances between likewise locus pairs separated by the same genomic distance but lying in the interior of a single domain. Error bars indicate 95% confidence intervals derived by resampling.
- Fig. 24M shows a standard deviation matrix of the inter-loci spatial distances for Chr21. For each pair of regions, the standard deviation of the distances between the corresponding pair of loci in all single chromosome copies is shown.
- Fig. 24N shows boxplots of the physical sizes of chromatin domains of different genomic sizes in Chr21, measured by the radii of gyration. For each genomic size, the median (center lines), 25th - 75th percentiles (boxes) and 10th - 90th percentiles (whiskers) are shown.
- Chromatin compartments in single chromosomes were used to examine how chromatin loci in A and B compartments are arranged in single cells.
- the ensemble A/B compartment boundaries were determined using principal component analysis (PCA) of the Pearson correlation matrix of the proximity frequency map for Chr21, derived from the imaging data, using a previously described algorithm (Fig. 18A; Fig. 25A).
- PCA principal component analysis
- the compartment boundaries obtained from the imaging data were highly similar to those determined from previously published ensemble Hi-C data (Fig. 25A).
- the compartment boundaries obtained from the ensemble proximity frequency map were used to assign A/B identity for individual loci in individual cells.
- compartment- A loci and compartment-B loci organization in single chromosomes.
- a high degree of variation in the arrangement of A and B loci was observed between individual chromosome copies from cell to cell (Fig. 18B). While in some chromosomes, A and B loci were segregated into essentially non-overlapping spatial territories, other chromosomes exhibited substantial spatial overlap between A and B loci. Intriguingly, compartment A loci in the same chromosome were sometimes separated into multiple “micro-compartments”
- a and B loci were assessed for their degree of spatial segregation in individual chromosomes.
- a local-density-based approach was devised and for each imaged locus, the local density of other A and B loci was computed (Fig. 25B).
- compartment- A loci on average, tended to be surrounded by A loci, and the same was true for B loci (Fig. 25C).
- An A/B segregation score was further defined for each individual chromosome based on the purity of loci observed in the spatial volumes harboring the majority of A or B loci (Fig. 18C).
- Chr21 is one of the smallest chromosomes, with a size of only -48 Mb, and is partitioned into only a small number of contiguous A and B regions.
- Chromosome 2 Chromosome 2
- Chr2 was traced by labeling and imaging 50-kb segments at intervals of 250-kb along its genomic sequence. The same approach described above was used to call A and B compartments in the p and q arms of the chromosome based on the imaging data (Fig. 18D; Fig.
- Figs. 18A-18I show the compartment structure in single chromosomes and relationship between transcription activity and local chromatin content.
- Fig. 18A shows a Pearson correlation matrix for genomic-distance-normalized proximity frequencies of Chr21 derived from our imaging data. Two loci are considered in proximity if their distance is smaller than a cutoff distance of 500 nm. Two bars at the bottom shows the A/B calling derived from proximity frequency matrix (shown for A compartments and B compartments) and G-banding of each genomic locus in the chromosome.
- Fig. 18B shows 3D renderings of individual copies of Chr21 in single cells, with A and B loci shown as balls. Flexible lines connect adjacent loci in genomic sequence. The bar at the bottom shows the A/B calling of each genomic locus in the chromosome derived from proximity frequency matrix. Scale bar: 1 micrometer.
- Fig. 18C shows a distribution of the A/B segregation score for individual copies of Chr21.
- an A (or B) dense volume is defined for each chromosome by thresholding the local A (or B) density such that 2/3 of A (or B) loci were contained within the volume (note that the A and B dense volumes can overlap for chromosomes that show spatial overlap between A and B loci).
- the purity of loci in the A (or B) dense volume of the chromosome was defined as the fraction of all loci within the volume being A (or B) loci
- the A/B segregation score of a chromosome copy was defined as the mean purity of the A and B volumes.
- Fig. 18D shows a Pearson correlation matrix for genomic-distance-normalized proximity frequencies for the p and q arms of Chr2 derived from our imaging data, and corresponding A/B calling and G-banding, as in Fig. 18 A.
- Fig. 18E shows 3D renderings of individual copies of Chr2, as in Fig. 18B. Scale bar: 1 micrometer.
- Fig. 18F shows a distribution of A/B segregation scores for Chr2 in single cells, as in Fig. 18C.
- n -3,100 chromosomes.
- Figs. 25A-25G show ensemble A/B compartment analyses for Chr21 and Chr2.
- Fig. 25 A shows a compartment calling based on principal component analysis for Chr21.
- First principal component (PCI) calculated for the Pearson correlation matrices from genomic- distance-normalized proximity frequency derived from imaging (top) and ensemble Hi-C (bottom) experiments are shown, with PCI value > 0 corresponding to compartment A and PCI value ⁇ 0 corresponding to compartment B.
- Fig. 25B shows a 3D rendering of compartment-A loci (A loci) and compartment-B loci (B loci), and A/B density ratios in a single copy of Chr21.
- a loci compartment-A loci
- B loci compartment-B loci
- A/B density ratios in a single copy of Chr21.
- Left A and B loci of a representative copy of Chr21.
- a and B compartment calling from the ensemble proximity frequency map derived from imaging is shown at the bottom bar.
- Right the same chromosome but with each locus colored by its local A/B density ratios.
- Fig. 25C shows mean A and B density scores for each imaged locus in Chr21 averaged across all imaged cells.
- the bottom panel represents the A or B compartment calling of each locus from the proximity frequency map derived from imaging.
- Fig. 25D shows histograms of the distributions of A/B segregation scores for individual copies of Chr21 in cells in the G1 and G2/S phases of the cell cycle.
- Fig. 25E shows a proximity frequency matrix derived from imaging (left) and ensemble Hi-C contact matrix (right) for Chr2.
- the Hi-C contact matrix is binned at 50 kb, but only the contacts from the imaged segments (selected at 250-kb intervals) are shown.
- Fig. 25F shows the same PC analyses as in Fig. 25A, but for the p-arm (top) and q- arm (bottom) of Chr2.
- Fig. 25G shows the same mean A and B density analysis as in Fig. 25C, but for Chr2.
- oligonucleotide probes targeting the first intron of 86 of the genes harbored in Chr21 were designed and sequential rounds of hybridization were performed to image nascent RNA transcripts of these genes followed by chromatin tracing. Furthermore, to more accurately detect the spatial position of the genes, the 5-kb genomic loci centered around the transcription start site (TSS) of each target gene was imaged.
- TSS transcription start site
- RNA probe hybridization was performed without heat denaturing the double- stranded genome, and then the RNA molecules were digested using an RNase treatment (a step that was also included when imaging chromatin alone) before performing chromatin tracing.
- RNase treatment a step that was also included when imaging chromatin alone
- Crosstalk between the RNA and DNA signals was confirmed to be negligible using this strategy (Figs. 26A-26J).
- a subset of the imaged genes showed transcription activity in any individual cell (Fig. 18G). It was examined how transcriptional activity was correlated with the local chromatin environment. To characterize the local A/B chromatin content, the local density of nearby A and B loci were calculated for each gene and their ratio (referred to hereafter as the A/B density ratio) was used as a metric for the local enrichment of active chromatin. It was found that for -80% of the genes studied, when a gene was actively transcribing, the local A/B density ratio at its TSS was higher than when the gene was not firing (Fig. 18H).
- Fig. 18G shows a 3D rendering of a single copy of Chr21 shown together with the transcriptional bursts of the measured genes. Balls represent all detected nascent RNA bursts in this chromosome. Scale bar: 500 nm.
- Fig. 18H shows the change (measured in log difference) in the A/B density ratios at the transcription start sites (TSS) of the imaged genes between actively firing and non-firing states.
- TSS transcription start sites
- the median A/B density ratio is computed at its TSS in chromosomes where the gene is firing and in chromosomes where it is not firing.
- the log-difference of these values for the 84 genes imaged on Chr21 is rank-ordered according to the magnitude of change in their median A/B density ratio. 79% of the imaged genes exhibited an increase in the A/B density ratio when they are actively firing, compared to not firing.
- Fig. 181 shows the change (measured in log difference) in the firing rates of the imaged genes as the local environment of the TSS of the gene is changed from low (bottom quartile) to high (top quartile) A/B density ratios.
- the log-difference in firing rate for the 84 genes imaged on Chr21 is rank-ordered according to the magnitude of firing rate. 79% of the imaged genes showed a higher firing rate when their TSS is in the top quartile, compared to the bottom quartile, of A/B density ratios.
- Figs. 26A-26J show measurements for RNA and DNA FISH probe crosstalk.
- Fi.g 26A shows example cells with their nucleus marked by DAPI (top) and the fluorescent signal of the FISH probes targeting the nascent RNA of a gene (BRWD1) (bottom).
- Fig. 26B is the same as Fig. 26A but for a different gene (SCAF4).
- SCAF4 a different gene
- the staining of Fig. 26A and 26B follows the protocol described in the RNA FISH protocol described in the “Cell culture preparation and primary/encoding probe hybridization” section in Example 19.
- Figs. 26C and 26D are the same as Fig. 26A and 26B, respectively, except that the RNA FISH protocol was modified to include an additional RNase treatment step to remove the cellular RNAs prior to addition of the FISH probes.
- the cells in Fig. 26C and Fig. 26D were imaged under similar illumination conditions to Figs. 26A and 26B and their fluorescent signal is displayed with the same contrast as in Figs. 26A and 26B.
- Fig. 26E shows the number of spots per cell with a signal-to-noise ratio > 3 for untreated and RNase-treated cells across 5 measured genes.
- Fig. 26F shows example cells with their nucleus marked by DAPI (top) and the fluorescent signal of the probes targeting a genomic locus (chr21:15.2Mb- 15.25Mb)
- Fig. 26G is the same as Fig. 26F, but for a different locus (chr21:14.95Mb- 15Mb).
- Figs. 26F and Fig. 26G follows the protocol described in the DNA FISH protocol described in the “Cell culture preparation and primary/encoding probe hybridization” section in Example 19.
- Figs. 26H and 261 are the same as Figs. 26F and 26G, respectively, except that the DNA FISH protocol was modified to omit the heat denaturation step and hence to remove the accessible genomic DNA sites.
- the cells in Figs. 26H and 261 were imaged under similar illumination conditions to Figs. 26F and 26G and their fluorescent signal is displayed with the same contrast as in Figs. 26H and 261.
- Fig. 26J shows the number of spots per cell with a signal-to-noise ratio > 3 for the cells treated with the heat denaturation step and the cells for which this step was omitted.
- Figs. 19A-19H show the dependence of domain-domain interaction on their A/B composition and genomic distance.
- Fig. 19 A Left is a 3D rendering of a “mixed” chromatin domain containing both A and B loci, flanked by “pure” domains comprised of only B loci in a copy of Chr2 in a single cell. Scale bar: 500 nm.
- Right pairwise distance matrix for the same region displayed on the left. The bars on the bottom and left of the matrix display the A and B calling of loci and the outline highlights the boundaries of chromatin domains.
- A/B calling is determined from the ensemble proximity frequency map of Chr2.
- Fig. 19B is the same as in Fig. 19A but for two pure domains, one comprised entirely of A loci and one entirely of B loci, instead of a mixed domain.
- Fig. 19C is a distribution of the fraction of loci being A loci in single-cell chromatin domains in Chr2.
- Fig. 19D is a single-cell spatial distance matrices of two example copies of Chr2.
- the first and third panels show the matrix for two whole chromosomes, while the second and last panels show a zoomed-in matrix for the region highlighted in yellow in the first and third panels, respectively.
- the side bars show the A/B compartment calling from the ensemble proximity frequency map.
- Fig. 19E shows domain contact probabilities for domains of different A/B compositions in Chr2.
- X and Y axes represent the fraction of loci within a domain being A loci (0% corresponds to pure B domain and 100% corresponds to pure A domain).
- Two domains are defined to be in contact if their insulation score is ⁇ 2. See Example 19 for the calculation of the insulation score.
- Fig. 19F shows domain contact probability between two pure A domains (A-A), between two pure B domains (B-B), and between one pure A and one pure B domain (A-B) in Chr2, plotted as a function of the genomic distance between the two interacting domains.
- Fig. 19G is the same as Fig. 19E, but for domain pairs with genomic distances larger than 80Mb.
- Fig. 19H is the same as Fig. 19F, but restricted to domain pairs with a high degree of intermixing (as defined by a low insulation score ⁇ 1).
- Scale bar 500 nm.
- Genome-scale chromatin imaging The sequential imaging approach described above allowed a high-resolution view of chromatin in individual chromosomes to be obtained. This straight sequential imaging approach is well suited for imaging chromatin structures that are comparable to or smaller than the diffraction-limited resolution.
- the number of genomic loci imaged increases only linearly with the number of imaging rounds in this approach.
- genome-scale chromatin imaging because many genomic loci could be simultaneously resolved and localized in the nucleus, it was reasoned that a much more efficient, non-linear scaling of the number of imaged loci with the number of imaging rounds would be possible.
- each genomic locus was assigned a unique 100-bit binary barcode with a Hamming weight of 2, i.e. each barcode containing two “1” bits and 98 “0” bits (Fig. 20A).
- the bit values (“1” or “0”) in these barcodes determined the presence or absence of signal for each locus across sequential rounds of imaging.
- a subset was further selected to encode the targeted genomic loci and optimized assignment of barcodes, such that loci with a “1” bit in the same barcode position were maximally separated in genomic space (see Example 19).
- This strategy allowed detection errors caused by overlapping signals from nearby chromatin loci to be minimized.
- this design allowed detection errors to be identified and discarded and measurement accuracy to be further improved.
- the barcodes were physically imprinted onto the targeted genomic loci using a high- diversity library of encoding probes, each containing a target region for binding to one of the targeted loci and a readout sequence chosen from 100 pre-designed readout sequences (Fig. 20A).
- Each readout sequence corresponds to one of the 100 bits, and the encoding probe set for each genomic locus (-400 probes per locus) contains only two distinct readout sequences, corresponding to the two bits that read “1” in the barcode assigned to that locus.
- the barcodes imprinted on the chromatin loci were detected by sequential hybridization of fluorescently labeled readout probes, each complementary to one of the 100 readout sequences (Fig. 20A).
- the adaptor probe strategy described for high-resolution whole-chromosome tracing was also used here.
- Two distinct adaptor/readout probes were introduced per hybridization round and imaged in two color channels, such that 2 bits were read out in each hybridization round.
- the homolog identities of the imaged loci were further assigned using a clustering algorithm, exploiting the tendency of chromosomes to occupy distinct territories in each nucleus.
- 1,041 genomic loci were selected for imaging, each ⁇ 30-kb in size, uniformly covering the 22 autosomes and the X chromosome in IMR-90 cells. Another requirement was that each chromosome contained at least 30 targeted loci, hence the number of loci imaged per chromosome homolog ranged between 30 and 80 depending on the length of the chromosome.
- 1,041 genomic loci in -5,400 individual cells across 5 biological replicates were imaged with a detection efficiency of -80% for each locus, yielding -1700 chromatin loci detected in each cell (Figs. 20D-20E).
- Figs. 20A-20H show genome-scale chromatin imaging by massively multiplexed, combinatorial FISH.
- Fig. 20A shows an imaging scheme.
- the targeted genomic loci are assigned error-robust barcodes, e.g. a subset of 100-bit binary barcodes with a Hamming weight of 2 (i.e. two of the 100 bits reading “1”).
- the barcodes are imprinted onto the genomic loci with encoding oligonucleotide probes, which recognize the loci and associate two distinct readout sequences with each locus, corresponding to the two bits that read “1” in the barcode assigned to the locus. Each bit is uniquely assigned a readout sequence.
- Each locus is labeled by a total of 400 encoding probes, but only 4 are shown. Fluorescent readout probes complementary to the readout sequences are sequentially added and imaged, allowing the bits that read “1” at each locus and hence the barcode identity of that locus to be determined.
- Fig. 20B shows representative images from multiple imaging rounds in the nucleus of a single cell. Fluorescent signal of the chromatin loci from readout probes and signals of 4',6-diamidino-2-phenylindole (DAPI), used as a nuclear marker, are shown. Scale bar: 5 micrometer.
- DAPI 4',6-diamidino-2-phenylindole
- Fig. 20C shows zoom-in images of a small region (white box in B) centered around one chromatin locus across all imaging rounds. The locus identity is determined based on the two readout probes (1 and 13) that give signals. Scale bar: 300 nm.
- Fig. 20D is a 3D rendering of all detected chromatin loci (spheres) in a single IMR-90 cell, color-coded according to the chromosomes that they belong to (index for chromosomes shown below the image). Adjacent loci in genomic sequence are connected by a flexible line. -1000 genomic loci are imaged.
- Fig. 20E shows chromatin loci of the same cell as in Fig. 20D but with two homologs of the indicated chromosomes highlighted.
- Fig. 20F shows a median distance matrix computed from -5,400 single cells. For each pair of loci, the median of observed 3D spatial distances between the loci across all cells is presented.
- Fig. 20G shows example images showing the positions of multiple chromosomes territories in single cells. Chromosomes are coded as indicated and shaded areas represent the convex hull surrounding all imaged loci. Only one homolog is shown per chromosome for clarity.
- Fig. 20H shows spatial distance matrices for the same cells shown in Fig. 20G. The spatial distance between each pair of chromatin loci is shown. Chromosome order is as noted beneath the matrices, with the two homologs of each chromosome separately shown.
- Figs. 27A-27J show genome-scale imaging by combinatorial FISH: localization error, reproducibility, and comparison with Hi-C.
- Fig. 27A shows a distribution of the displacement between the localizations of genomic loci measured in the combinatorial imaging run and those of the same loci re-imaged individually using sequential hybridization after completing the combinatorial imaging. 10 genomic regions in Chr6 were re-imaged across -2000 cells. The median displacement is -50 nm.
- Fig. 27B shows a proximity frequency matrix for all 1,041 genomic loci imaged by combinatorial FISH.
- the proximity frequency between a pair of loci was calculated as the number of incidences in which the measured distance between the loci is smaller than a cutoff distance of 500 nm divided by the total number of measured distances between the two loci.
- Fig. 27C shows a correlation plot for the proximity frequencies between pairs of loci within chromosomes derived from our imaging data and the number of contacts derived from ensemble Hi-C experiments, binned at 500 kb and centered around the target loci.
- the Pearson correlation coefficient is 0.91.
- the available Hi-C data for IMR90 cells is sparse for trans-chromosomal contacts, precluding a reliable comparison of trans-chromosomal interactions between our imaging data and the Hi-C data.
- Fig. 27D shows the correlation of pairwise distances between chromatin loci observed in two independent biological replicates of the genome-scale imaging experiments.
- the Pearson correlation coefficient between replicates is 0.98.
- the upper right cloud represents the /ra/7.s-chromosomal pairwise distances and the lower-left cloud represents the intra- chromosomal pairwise distances.
- Hi-C calling was used to classify the A/B compartment identity of the imaged loci because of the higher genomic resolution of the ensemble Hi-C data. 38% of the imaged loci belonged to compartment A, while 62% belonged to compartment B. To examine whether the extent of /ran.s-chromosomal interactions differs for active and inactive chromatin, the genomic loci in the /rani-chromosomal proximity frequency matrix were rearranged, placing all A loci next to each other followed by all B loci.
- compartment- A locus had on average a stronger tendency to interact / ra nv - c h ro m o s o m ally with another compartment-A locus than with individual compartment-B loci (Figs. 21A-21B), consistent with previous observations of /ran.s-chromosomal interactions between active chromatin.
- compartment-B loci showed comparable or lower /ran.s-chromosomal affinity towards each other than towards compartment-A loci (Figs. 21A-21B).
- trans- chromosomal A-A interactions appeared with a substantially stronger tendency than A-B interactions, which in turn appeared with a slightly stronger tendency than B-B interactions.
- the trans A/B density ratio The ratio of these two densities was determined (referred to hereafter as the trans A/B density ratio) (Figs. 21D-21E). This quantity provided a measure of the local enrichment of /ran.s-chromosomal active chromatin near the locus. It was noted that the majority (62%) of the imaged loci belonged to the B compartment, creating an overall bias for the A/B ratio to be smaller than 1.
- Figs. 21A-21E show enrichment of active-active chromatin interactions in trans- chromosomal interactions.
- Fig. 21 A shows normalized /rani-chromosomal proximity frequency matrix. The proximity frequency between each /ran.s-chromosomal locus pair (pair of loci on different chromosomes) is shown, with pairs of loci considered to be in proximity if their distances are smaller than a cutoff distance of 500 nm. The loci are reordered such that compartment-A loci appear first, followed by compartment-B loci, hence the top left block represents interactions between pairs of A loci and the bottom right represents interactions between pairs of B loci.
- Fig. 21C shows the median proximity frequency between pairs of chromatin loci within the same chromosomes as a function of their genomic distance, averaged for pairs of loci separated by the same genomic distance across all chromosomes. Median contact frequencies are shown for pairs of A loci (A-A), pairs of B loci (B-B) and for mixed pairs of A and B loci (A-B).
- Fig. 2 ID shows distributions of compartment-A and compartment-B loci in two single cells.
- the left panels represent the locations of all detected loci within a single z-plane in a single nucleus, with compartment-A loci and compartment-B loci.
- the shading of each locus represents the ratio of the local densities of /rani-chromosomal A and B loci, i.e. the trans A/B density ratio, in accordance with the scale bar shown on the right.
- Fig. 27E are bar plots for the percentage of loci whose A/B compartment assignment agree between the genome-scale imaging data and Hi-C data for each human autosome. On average, -81% of the loci in each chromosome showed agreement in A/B assignment between our imaging data and the Hi-C data.
- Fig. 27F shows the median normalized /rani-chromosomal A-A, A-B, and B-B proximity frequencies (defined as in Figs. 21 A and 2 IB) as a function of the cutoff distance used to evaluate proximity.
- the normalized proximities are calculated from -5,400 IMR-90 cells.
- the median normalized trans-chromosomal A-A, A-B, and B-B proximity frequencies after perturbing the loci positions with a 3D Gaussian noise term with a standard deviation of 50 nm, comparable to the estimated localization measurement error, as shown in Fig. 27A.
- Fig. 27G is the same as Fig. 27F, but when additional data from alpha-amanitin treated cells is pooled with the untreated cells (for a total of -9,500 cells) alpha-amanitin treated cells showed similar enrichment of /ran.s-chromosomal A-A over A-B and B-B proximity frequencies, as untreated cells (Figs. 29A, 29B). This pooling result suggests that the lower enrichment for A-A interactions observed at lower cutoffs distance in Fig. 27F is likely a result of poorer statistics with a lower number of cells.
- Fig. 27H shows the median normalized /ran.s-chromosomal A-A, A-B, and B-B proximity frequencies, as a function of the number of cells included in the analysis. Cells were subsampled randomly from the -5,400 untreated IMR-90 cells imaged and the proximity cutoff distance was fixed to 500 nm.
- Figs. 29A-29F show the effect of transcriptional inhibition on the trans-chromosome chromatin interactions and the nuclear body association rates of chromatin loci.
- Fig. 29A shows normalized /ran.s-chromosomal proximity frequency matrix, as in Fig. 21 A, but for cells treated with alpha-amanitin to inhibit transcription.
- Fig. 29B shows a distribution of normalized /ran.s-chromosomal A-A, B-B and A-B proximity frequencies shown as box plots, as in Fig. 21B, but for cells treated with alpha- amanitin.
- the normalized /ran.s-chromosomal A-A, B-B and A-B proximity frequencies for untreated cells from Fig. 2 IB is reproduced here.
- Fig. 29C shows distributions of the local trans A/B density ratio across imaged A and B loci, as in Fig. 2 IE, but for cells treated with alpha-amanitin.
- the histogram represents a randomization control where the A and B compartment identity is randomly shuffled, while keeping the total number of A loci and the total number of B loci unchanged.
- Multi-modal imaging of chromatin, nascent RNA and nuclear structures To place the 3D organization of chromatin in the context of its functional activity and other nuclear structures, the combinatorial imaging method was expanded to allow simultaneous measurements of chromatin organization together with transcriptional activity of the imaged genomic loci, as well as nuclear landmarks in single cells. Specifically, the aforementioned 1,041 genomic loci together with the nascent RNA transcribed from each of the 1,137 genes located at these loci were imaged and simultaneously with important nuclear structures, including nuclear speckles and nucleoli (Fig. 22A).
- RNA and nuclear- structure imaging within the same cells multiplexed imaging of the intronic RNAs of the 1,137 genes was performed, by adopting a similar combinatorial imaging strategy to the one described above for chromatin (Fig. 22 A).
- the RNAs were encoded with a 54-bit, Hamming weight 2 code, and 1,137 of the possible barcodes to encode the genes were selected, in a way similar to how the barcodes for chromatin imaging were selected to minimize the chance of imaging spatially proximal genes in the same bit.
- RNA transcripts were enzymatically digested (a step also carried out in the single-modal chromatin imaging experiments) and multiplexed DNA FISH was performed as described above to image the 1,041 genomic loci (Fig. 22A).
- Decoding of genomic loci and nascent RNA transcripts was performed largely independently, with the additional constraint for the transcripts to colocalize with their harboring genomic loci (See Example 19). This procedure further improved detection accuracy for RNA transcripts and allowed the estimation of the detection efficiency (-90%) for the transcription bursts at each genomic locus (see Example 19).
- nuclear speckles and nucleoli were imaged, using immunofluorescence against known molecular components of these structures (Fig. 22 A).
- the fluorescent signals for nuclear speckles and nucleoli displayed a high signal- to-noise ratio (>25) even with immunofluorescence staining performed after DNA FISH.
- the positions of nuclear lamina were estimated by computing a convex hull surface encompassing all imaged genomic loci. Together, these multi-modal measurements allowed an integrated single-cell view of 3D genome structure, transcriptional activity and nuclear organization (Fig. 22B). These multi-modal imaging experiments were performed on -3700 individual cells, in two biological replicates. Chromatin imaging data from these multi modal experiments were also included in the 5 replicates and -5,400 cells described above for 3D genome organization analyses.
- Figs. 22A-22J show multi-modal genome-scale imaging of chromatin and transcription activity in the context of nuclear structures.
- Fig. 22A top: Illustration of the multi-modal imaging scheme that combines chromatin (left panel), nascent RNA transcripts (middle panel) and nuclear bodies (right panel) imaging to generate an integrated view of chromatin organization in the context of nuclear structures and functional activity.
- -1000 genomic loci, nascent RNA transcripts of -1100 genes in the targeted loci, and two types of nuclear bodies (nuclear speckles and nucleoli) are imaged.
- Fig. 22B is a 3D rendering of chromatin loci, transcriptional bursts and nuclear bodies in a single cell.
- Left All detected chromatin loci, coded by chromosome (based on the chromosome index shown below). Middle: All detected intronic RNAs shown as colored spheres, with shadings indicating the identities of the imaged genes and sphere size representing transcription burst size.
- Right Volume-filling representations of detected nuclear bodies.
- the nuclear lamina is identified as the surface of the convex hull surrounding all detected chromatin loci (shaded gray area).
- a chromatin locus is considered associated with nuclear lamina or a nuclear speckle if the distance of the locus to the nuclear periphery or the nearest speckle is ⁇ 250 nm.
- the values of trans A/B density ratio shown are the median values across all imaged cells.
- Fig. 221 shows the association frequency with nucleoli for all imaged genomic loci, ordered by genomic position. Black vertical lines indicate the locations of centromeres and brackets highlight chromosomes containing ribosome-encoding genes (rDNAs).
- Fig. 22J shows the correlation of transcription with nuclear structure association. Circles are the fold-change in the transcriptional burst frequency for individual genomic loci when comparing the populations of cells in which the locus is lamina associated versus non- lamina-associated (left) and speckle- associated versus non-speckle associated (right). The dotted line highlights no change and the solid lines represent the median fold-change in each case.
- Figs. 271 and 27J show a correlation between replicates of RNA imaging for each gene’s burst frequency (Fig. 271) and burst size (Fig. 27J). Pearson correlation coefficients are 0.94 and 0.81, respectively.
- Figs 29D and 29E show representative images of individual nuclei with imaged chromatin loci, nucleoli, and nuclear speckles shown for untreated cells (Fig. 29D) and cells treated with alpha-amanitin (Fig. 29E).
- Fig. 29F shows a fold change in the rate of associate of each locus with lamina (left) and nuclear speckles (right) upon alpha-amanitin treatment.
- the data point for each genomic WO 2021/138078 - Ill - PCT/US2020/065797 locus is shown in circles, the solid lines are the median fold changes of all loci in each case, and the dotted line represents no change. It is noted that the nuclear volume and the size and number of nuclear speckles also changed upon treatment with alpha-amanitin, and these might partially contribute to the changes in nuclear body association.
- the left panels show example images displaying A loci and B loci in a single z-plane of single cells.
- the right panel shows the distribution of distances to the nuclear periphery for A loci and B loci in these single cells.
- the nuclear periphery is identified as a convex hull surrounding all detected chromatin loci.
- Figs. 30A-30D show enrichment of /ran.s-chromosomal active chromatin interactions in different nuclear environments.
- Fig. 30A shows normalized /rani-chromosomal proximity frequency matrix, as in Fig. 21 A, but considering only loci that are not associated with nuclear speckle. For each locus pair, only cells in which neither locus is associated with nuclear speckles are considered.
- Fig. 30B shows tran.s-chromosomal proximity frequency for pairs of A loci (A- A), pairs of B loci (B-B), and pairs comprised of one A and one B locus (A-B), as in Fig. 21 5B, but considering only the cells in which neither locus is associated with nuclear speckles.
- Fig. 30C is the same as Fig. 30A, but for pairs of lamina-associated loci. For each locus pair, only cells in which both loci are associated with nuclear lamina are considered.
- Fig. 30D is the same as Fig. 30B, but for pairs of lamina-associated loci. For each locus pair, only cells in which both loci are associated with nuclear lamina are considered.
- trans A/B density ratios were calculated and the median values of this quantity were determined for two populations of cells (determined independently for each genomic locus): (i) the cells where the locus under consideration exhibited transcriptional activity, and (ii) the cells where the locus appeared transcriptionally silent (Fig. 23A).
- a consistent trend for a higher trans A/B density ratio was observed when the locus was actively transcribed: 86% of the imaged loci exhibited a greater trans A/B density ratio when in the actively transcribing state as compared to the silent state (Fig.
- Figs. 23A-23D show the correlation between transcriptional activity and local enrichment of /ran.s-chromosomal active chromatin.
- Fig. 23A shows single-cell images of chromatin loci and transcriptional activities. Left: Locations of all imaged A and B loci in a single z-plane from a single nucleus. Middle: Local trans A/B density ratios for the same loci, coded based on the scale bar. Right: Same as the middle panel, with detected transcriptional bursts overlaid and displayed as circles. Scale bar: 3 micrometer
- Fig. 23B shows the change (measured in log difference) in the trans A/B density ratios for each imaged locus between actively firing and non-firing states.
- the trans A/B density ratio was calculated for the cells in which the genomic locus was actively transcribed (designated as transcribed) and for the cells in which it was not transcribed (designated as silent).
- the log-difference of the medians of these values for each imaged locus was rank-ordered according to the magnitude of change. 86% of the imaged loci exhibited an increase in the A/B density ratio when they are actively firing, compared to not firing.
- Fig. 23C shows the change (measured in log difference) in the firing rates of the imaged genes between cells in which the trans- A/B density ratio at the locus harboring the gene changes from low (bottom quartile) and high (top quartile).
- the log-difference in firing rate for all genes imaged was rank-ordered according to the magnitude of firing rate. 89% of the imaged genes showed a higher firing rate when their harboring locus was in the top quartile, compared to the bottom quartile, of trans- A/B density ratios.
- Fig. 23D shows swarm plots showing the fold change of local trans A/B density ratios between transcribed and silent states for the imaged gene-containing loci, conditioned on their nuclear body association status.
- the fold change was computed in the trans A/B density ratio between transcribed and silent states of the locus, considering, from left to right, respectively: all cells, only the cells in which the locus was associated with a nuclear speckle, only cells in which the locus was associated with the lamina, and only cells in which the locus was not associated with a nuclear speckle nor with the lamina (empty circles).
- the median trans A/B density ratio in each state was determined for each locus and each association condition, and the log2 of the fold change between the two states is shown.
- the dotted line represents no change and the solid lines represent the median fold change across all loci in each case.
- Chromosome-wide and genome-scale chromatin imaging Reported herein is massively multiplexed chromatin imaging for determining the 3D conformation of chromatin across multiple scales of genome organization in single cells. The ability to image >1000 genomic loci in thousands of individual cells was demonstrated. The approach further allowed the placement of the 3D chromatin organization in its native functional and structural context by combining chromatin tracing with nascent-transcript and nuclear- structure imaging, and the ability to simultaneously image >1000 genomic loci, the transcription activity of >1000 genes residing in these loci, as well as landmark nuclear structures, including nuclear speckles and nucleoli was demonstrated.
- the high-throughput imaging technology shown in this example has several advantages for studying chromatin organization.
- Second, the method is intrinsically a single cell approach and can reveal detailed chromatin structures in individual cells.
- the high (nearly 100%) detection efficiency of individual chromatin loci by the imaging methods allows a high capture rate of pair-wise chromatin interactions, which can provide a high- definition view of chromatin structures in single cells.
- the chromatin tracing technology can be readily combined with other imaging modalities. This includes multiplexed transcriptional imaging and nuclear structure imaging as demonstrated in this study, but could also be further expanded to include other modalities such as imaging of epigenetic modifications or the degree of chromatin accessibility. Such multi-modal imaging can provide key insights into the relationships between chromatin structure, nuclear organization, and transcriptional activity.
- loci were targeted uniformly across chromosomes to provide an unbiased view of the overall 3D chromosome and genome organization, this method could also be used to target genomic loci with specific structural and functional properties.
- An interesting direction would be to target loci that either contain specific genes or regulatory sequences, or are bound by specific nuclear architecture proteins, such as CTCF or cohesin, to study the interactions between these loci and their relationship with transcription.
- a large set of potential promoters and enhancers can be targeted, and their interactions can be studied while simultaneously imaging the transcription activity of the genes governed by the promoters in the same cells.
- IMR-90 cells were purchased from American Type Culture Collection (ATCC, CCL-186) and grown according to the recommended protocol.
- Oligonucleotide probe design Choice of target genomic regions. For high-resolution whole-chromosome imaging by sequential hybridization, the target chromosome were first partitioned into 50-kb segments. After screening out repetitive elements and regions where ⁇ 100 unique probes can be designed per 50-kb segments, a total of 651 target genomic loci were kept for Chr21 and 4,500 target genomic loci for Chr2. Primary probes were then designed for each 50-kb segment (-500 oligonucleotide probes) and the 350 most centrally positioned probes per segment were kept for sequential imaging. For Chr21 imaging, all 651 genomic loci were imaged. For Chr2 imaging, 250-kb genomic resolution was aimed for, and hence only designed primary probes for one in every five 50-kb segments.
- RNA transcripts on Chr21 For imaging nascent RNA transcripts on Chr21, genes were selected for which >50 primary probes (see “Primary/encoding probe design” section, below) could be designed on their first introns from all the protein-coding genes on Chr21. A total of 86 genes that are interspersed across Chr21 were selected. In order to facilitate the accurate detection of the spatial positions of transcription initiation events, probes to target a 5-kb segment of DNA around the transcription start site (TSS) of each gene were designed. For genome-scale chromatin imaging by the combinatorial imaging strategy, genomic loci were chosen for imaging in the following way. For each human chromosome (except the Y chromosome), a 30-kb segment every ⁇ 3 Mb of spacing was selected.
- RNA transcripts For imaging of nascent RNA transcripts in the genome-scale imaging, all intron- containing genes that completely or partially overlapped with the 1,041 targeted genomic loci were chosen. Encoding probes for the introns of all of these RNAs were chosen, such that each RNA had -20 encoding probes and that the targeting sequences of the encoding probes were kept as close as possible to the transcription start site. A total of 1,137 genes were targeted.
- Barcode design for genome-scale imaging by combinatorial FISH Barcode design for genome-scale imaging by combinatorial FISH.
- Binary barcodes for imaging the 1,041 genomic loci were chosen in the following fashion. First, all possible 100-bit binary barcodes with a Hamming weight of 2 (i.e. each barcode containing two “1” bits and 98 “0” bits) were generated and 1,041 barcodes from this list were randomly selected. The selected barcodes were then arbitrarily assigned to the 1041 genomic loci first. Next, barcodes were exchanged randomly between the used and unused code pool, as well as between loci from different chromosomes, in order to minimize, for each chromosome, the variance in the number of loci appearing (i.e. reading “1”) across different bits.
- loci within the same chromosome were allowed to exchange barcodes and the largest minimal genomic distance between loci with barcodes reading “1” at the same code position were optimized.
- code assignments with identical minimal genomic distances the one that minimized the coefficient of variation of genomic distances was selected (so that genomic distances have both larger means and smaller standard deviations).
- Barcodes for imaging the nascent RNA transcripts of the 1,137 genes were chosen similarly, but using a 54-bit, Hamming distance 2 code instead of a 100-bit, Hamming distance 2 code.
- Primary/encoding probe design Primary/encoding probes for chromatin imaging were synthesized from a pool of oligonucleotides purchased from Twist Biosciences. Each oligo in this pool used the following sub-sequences (from 5’ to 3’): a 20-nucleotide (nt) or
- RT forward priming region for PCR amplification and reverse transcription
- 20-nt readout sequence corresponding to the genomic locus targeted by the probe in the case of sequential imaging or one of the bits in which the genomic locus targeted by the probe will be imaged in the case of combinatorial imaging
- 42-nt or 40-nt target sequence for sequential or combinatorial imaging, respectively
- a 20-nt readout sequence designed to bind uniquely to a single targeted genomic locus, an additional 1-2 copies of the 20-nt readout sequence described above, and a
- RNA imaging Similar designs with minor modifications were used for nascent RNA imaging.
- the forward and reverse priming sequences were chosen from a previously generated list of random 20-nt sequences optimized for PCR, as described previously.
- the readout sequences were chosen via the following process. First, a list of 30-nt sequences with minimal homology to the human genome was created, as previously described. Then, a subset of these sequences was ranked by observed signal to noise ratio (SNR) and the top 100 were chosen as DNA readout probes. For sequential imaging, substantially more readout sequences were needed due to the larger number of hybridization rounds. Hence, the same procedure outlined previously was followed to select -1,200 candidate readout sequences. Then these candidates were filtered to ensure a GC content of 40-60% and a melting temperature of 57-67 degrees Celsius. These sequences were further filtered using BLAST such that no readout sequence had hits with HSP score larger or equal to 17. Lastly, the readout sequences were chosen by reverse-complementing the last 20-nt of each of these sequences.
- SNR signal to noise ratio
- the 42-nt or 40-nt target sequence was chosen similarly to a procedure described previously. Briefly, the following procedure was repeated for each genomic region of interest (see the “Target genomic regions” section above). First, a list of all 42-nt or 40-nt sequences complementary to the genomic region of interest was created (starting at each possible base in the targeted region). Then, sequences were filtered by requiring them to be within a defined range of melting temperatures and GC content. The remaining sequences were then further filtered by limiting the allowed degree of homology to the human genome, the human transcriptome and a database containing repetitive sequences using the same procedure as previously.
- sequences used for whole-chromosome imaging by sequential hybridization had an additional filtering step using BLAST in which each target sequence was ensured to match uniquely the intended genomic locus. Finally, target sequences were selected from the remaining sequences after the final filtering step such that no genomic overlap exists between any pair of target sequences.
- each target sequence was concatenated to two identical copies of the readout sequence assigned and then concatenated to the forward and reverse PCR primers.
- each of the chosen 40-nt target sequences for each target genomic locus was altematingly assigned to 2 groups spanning the entire target locus. Each of these groups was associated with a single readout sequence, corresponding to one of the two bits in which the locus would be imaged. Then, each target sequence was concatenated to two identical copies of the readout sequence assigned to its group, and then concatenated to the forward and reverse PCR primers.
- Probes for RNA imaging were designed similarly, with the exception that they contained 3 copies of an identical readout sequence on every probe, one at the 5’ end and two at the 3’ end of the target region. Readout sequences for RNA imaging were orthogonal to those used for DNA imaging and were selected from the same ranked list of tested readout sequences.
- Microscope setup for image acquisition was performed using a custom-built microscope system.
- the system was built around a Nikon Ti-U microscope body with a Nikon CFI Plan Apo Lambda 60x oil immersion objective with 1.4 NA.
- Illumination was based on one of two alternatives: solid-state, single-mode lasers with the following wavelengths: 405 nm (Coherent, Obis 405 nm LX 200 mW), 560 nm (MPB Communications, 2RU-VFL-P-2000-560-B1R), 647 nm (MPB Communication, 2RU-VFL- P-1500-647-B1R) and 750 nm (MPB Communication, 2RU-VFL-P-500-750-B1R).
- 405 nm Coherent, Obis 405 nm LX 200 mW
- 560 nm MPB Communications, 2RU-VFL-P-2000-560-B1R
- 647 nm MPB Communication, 2RU-VFL- P
- the output of the 560-nm, 647-nm and 750-nm lasers were controlled by an acousto optic tunable filter (AOTF) while the 405-nm laser was controlled directly via its laser control box.
- AOTF acousto optic tunable filter
- a custom dichroic (Chroma, zy405/488/561/647/752RP-UFl) and emission filter (Chroma, ZET405/488/461/647-656/752m) were used to separate excitation and emission illuminations.
- a Lumencor CELESTA light engine (a fiber-coupled solid-state laser based illumination system) with the following wavelengths: 405 nm, 446 nm, 477 nm, 520 nm, 546 nm, 638 nm and 749 nm.
- This system was used with a penta-bandpass dichroic (IDEX, FF421/491/567/659/776-Di01-25x36) and a penta-bandpass filter (IDEX, FF01- 441/511/593/684/817-25).
- IDEX penta-bandpass dichroic
- IDEX penta-bandpass filter
- the illumination was flattened using a Refractive Beam Shaper (Newport Optics, GBS-AR14) or a vibrating optical fiber (Errol, custom Albedo unit).
- CMOS camera Hamamatsu FLASH4.0 or Hamamatsu C 13440 with factory calibration for single-molecule imaging
- Sample position in three dimensions was controlled using a XYZ stage (Ludl).
- a custom-built auto focus system was used to maintain a constant focal plane over prolonged periods of time.
- NI PCIe-6353 National Instruments Data Acquisition card
- custom software see “Software for controlling experimental components” below.
- the fluidics system used several main components: a pump, a set of valves connected in series, a flow chamber in which the sample was mounted, and tubing and connectors.
- a peristaltic pump (Gilson, MINIPLUS 3) was used to generate flow in the system.
- the pump was connected to an array of 8-way valves (Hamilton, MVP and HVXM 8-5), connected in series. In this study, 3-5 valves connected in such a manner were used. Each valve’s last connection was used as the input of the next valve in the series (except for the last one), while the rest were connected to a tube containing the buffer for a single round of hybridization.
- valves were used for imaging, bleaching and wash buffers (see “Experimental procedures and protocols” section).
- This valve system was used to flow the various buffers into the flow chamber (Bioptechs, 060319-2), in which the sample was placed.
- the chamber output was connected to a waste collection vessel, forming an open flow system.
- Components were connected using elastic plastic tubing, and connections were sealed using a pressure adhesive (Blu-tack).
- the system was controlled using a custom software (see “Software for controlling experimental components” below). Overall, this system allowed for 20-36 rounds of hybridization (depending on the number of valves and the number of spots reserved for special buffers).
- the buffers were replaced with new ones via the following procedure: the output from the valve system was directly connected to the waste collection vessel, bypassing the sample-containing chamber. Then all valves were washed using 30% formamide and double-distilled water. Next, the new set of buffers was introduced, and the chamber was reconnected to the flow system. Lastly, the experiment resumed for the next round of hybridization.
- Hal was the software package used to control and synchronize all illumination and microscope components. It was noted that in some cases it is necessary to write drivers for components, which are not included in this package. Hal is also used to define imaging parameters, such as illumination strength, sequence of stage and illuminations operations during imaging (e.g. during a z-scan), exposure time etc.
- Step which was a module used to take mosaic images (i.e. a composite image made up of many individual fields of view) and select regions for imaging in experiments.
- the general flow of an experiment is that, before the experiment starts, Hal and Kilroy are loaded with the parameters and specifications to be used. After the sample is loaded and the chamber is filled with imaging buffer, a mosaic image of the DAPI channel is taken using Steve, and regions of interest are selected. A file is then generated to specify the sequence of operations throughout the entire experiment and is loaded to Dave, together with the coordinates of the selected regions of interest. The rest of the experiment is run automatically, without manual intervention. If the number of rounds in the experiment exceeds the capacity of the flow system, the automatic sequence specifies actions up to the capacity of the system. The buffers are then replaced (see “Fluidics system configuration” section above), a new Dave file is created, and this is repeated until all rounds of imaging are completed.
- Primary/encoding probe synthesis Primary/encoding probes were amplified from the template library described above (see “Primary/encoding probe design” above). This was done using a previously described amplification protocol involving the following steps: first, the initial oligo pool was expanded using limited-cycle PCR for approximately 20 cycles.
- the reverse primer used in this step also introduced a T7 promoter sequence via primer extension.
- the resulting product was purified via column purification and underwent further amplification and conversion to RNA by a high-yield in-vitro transcription reaction.
- the RNA product was converted back to single-stranded DNA by a reverse transcription reaction.
- the product of the previous step was subjected to alkaline hydrolysis (to remove residual RNA) and column purified (DNA Clean & Concentrator Kit, Zymo Research D4003 and D4033).
- the product of the previous step was dried in vacuum and resuspended in water to achieve the desired concentration of primary probe. All primers were purchased from Integrated DNA Technologies (IDT).
- HC1 hydrochloric acid
- PBS 0.1 M hydrochloric acid
- SSC 2x saline-sodium citrate buffer
- AM9342 50% formamide
- the cell coverslip was inverted and placed on a drop of 50 microliters of hybridization buffer (2x SSC, 50% formamide, 10% dextran sulfate (Sigma-Aldrich, D8906) containing a mixture of primary/encoding probes at ⁇ 25 micromolar total concentration with or without 10 microgram Human Cot-1 DNA (ThermoFisher, 15279011)) in a 60-mm petri dish.
- the dish was partially submerged in a water bath at -90 °C for 3 minutes and incubated at 47 °C in a humidified chamber for 16-36 hours.
- the sample was washed in 2x SSC and 40% formamide for 30 minutes and post-fixed with 4% PFA in 2x SSC for 10 minutes at room temperature.
- the sample was then incubated for 2-3 minutes with fiducial beads (either ThermoFisher F8805 or ThermoFisher F8792) in 2x SSC and stained with 1 micromolar 4’,6-diamidino-2-phenylindole (DAPI; ThermoFisher D1306) in 2x SSC for 5-10 minutes, and then stored in 2x SSC until imaging.
- fiducial beads either ThermoFisher F8805 or ThermoFisher F8792
- RNA staining was identical to the above-described protocol up to treatment with HC1. After this step, cells were incubated in pre-hybridization buffer for 10 minutes, and the cell coverslip was then inverted and placed on a drop of hybridization buffer containing primary/encoding probes targeting the RNA introns at -1 micromolar total concentration, as described for DNA staining. In this case, however, no 90 °C heat denaturation was performed, and cells were immediately incubated at 47 °C in a humidified chamber for 16-36 hours.
- RNAse inhibitor either NEB M0314 or Fisher Scientific N2615
- the sample was washed in a formamide solution and post-fixed with PFA as described for DNA above. It was then incubated with fiducial beads and stained with 1 micromolar DAPI, before being stored in 2x SSC until imaging. After RNA imaging, the sample was removed from the microscope, the cells were treated with RNase A and then the DNA hybridization proceeded in the same manner as described above for DNA imaging without RNA imaging.
- each round of hybridization used the following general steps: first, the hybridization buffer was flowed in with a set of oligonucleotide probes specific to each round, as described below. Then, it was incubated for 10 minutes at room temperature. Next, the wash buffer was flowed through, and it was incubated for -200 seconds, and lastly, the imaging buffer was flowed through.
- Imaging buffer was prepared as described previously, and used 60 mM Tris pH 8.0, 10% w/v glucose, 1% Glucose Oxidase Oxygen Scavenger Solution (containing -100 mg/mL Glucose Oxidase (Sigma-Aldrich, G2133) and a 1:3 dilution of catalase (Sigma- Aldrich, C3155)), 0.5 mg/mL 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox; Sigma-Aldrich, 238813) and 50 mM Trolox Quinone (generated by UV irradiation of a Trolox solution). Trolox was dissolved in methanol before being added to the solution. After preparation, the imaging buffer was covered by a -0.5 cm thick layer of mineral oil to prevent exposure to oxygen.
- the hybridization buffer and wash buffer were made up of 35% and 30% formamide in 2x SSC, respectively, with the hybridization buffer also containing 0.01% v/v Triton-X.
- the hybridization buffer was kept separately for each hybridization round and contained two (for genome-scale chromatin imaging by combinatorial FISH) or three (for whole- chromosome imaging by sequential hybridization and for genome-scale chromatin and RNA imaging by combinatorial FISH) sets of readout probes. Fluorescent signal was introduced in the following ways: for whole-chromosome imaging by sequential hybridization, the hybridization buffer contained three fluorescent readout probes (Alexa750, Alexa647 or Cy5, and Cy3) added at 30 nM concentration.
- Fluorescent readout probes contained a disulfide bond linking the fluorophore to the oligonucleotide, as described previously, to allow efficient removal of signal between rounds.
- hybridization buffer included two fluorescent readout probes, one labeled with Cy5 or Alexa647 and the other labeled with Alexa750.
- Fluorescent readout probes used either: 1) a fluorescently labeled oligo complementary to a readout sequence common to all encoding probes imaged in a given bit, added at 100 nM concentration, or 2) a combination of an adaptor oligo having the sequence complementary to a readout sequence, concatenated to an additional common readout sequence (common to all adaptors in each color channel), as described above, and a fluorescently labeled readout probe complementary to this common readout sequence.
- the adaptor and common readout probes were pre-mixed in a 1:1.5 ratio and added to a final concentration of -100 nM.
- the adaptor and readout probes were hybridized sequentially to the sample.
- each round contained three adaptor probes, one for each color channel, as described above.
- Each round included two discrete hybridization steps - first the adaptors were flowed in, hybridized, and excess material was washed. Then three fluorescent readout probes, complementary to the common readout sequences on the adaptors, respectively labeled with Cy3, Cy5 (or Alexa647) and Alexa750, were flown in sequentially. After the fluorescent readouts were hybridized, imaging buffer was flowed in and signal was collected.
- a round of imaging was performed to acquire the DAPI signal and identify nuclear boundaries.
- 651 genomic loci on Chr21 over -220 rounds or 935 genomic loci on Chr2 over -320 rounds were imaged, all in 3 color channels.
- genome-scale chromatin imaging by combinatorial FISH the entire set of 1,041 genomic loci were imaged in 50 rounds of hybridization and 2 color channels per round. In each round, the genomic loci were imaged in 3D by stepping the stage in the z-dimension.
- Nascent RNA transcripts for the 86 genes on Chr21 were imaged sequentially over 31 rounds in 3 colors, and the genome- scale imaging of the RNA transcripts of 1,137 genes were imaged in 3D in 18 rounds in 3 colors.
- the TSS of the 86 genes over 29 rounds was imaged in 3 colors. Additional rounds were used to relabel sets of genomic loci and assess chromatic aberration and bleedthrough between color channels, as well as stability of the sample and imaging instrument. Imaging of -60 fields of view containing a total of -1,000- 3,000 cells took -12-18 days for sequential imaging of whole-chromosome imaging by sequential hybridization and 3 days for genome- scale chromatin imaging by combinatorial FISH.
- the 3-5 valve system allowed loading up to 20-36 different hybridization solutions.
- a gentle post-fixation step was performed with 2% PFA in 2x SSC for 5 minutes periodically (every ⁇ 4 days) to maintain the structural integrity of the sample.
- Antibody labeling and imaging was performed immediately after RNA or DNA imaging. Following completion of imaging via the protocols described above, samples underwent the following steps: Samples were incubated with blocking solution (PBS with 0.1% v/v Tween-20 (Sigma-Aldrich P9416) and 1% w/v bovine serum albumin (BSA; Jackson Immunoresearch 001-000-162)) for 30 minutes. Samples were incubated with primary antibody diluted in blocking solution for 1 hour. Samples were washed 3 times in PBS with 0.1% Tween-20 for 5 minutes each. Steps 2 and 3 were repeated for a fluorescently tagged secondary antibody.
- blocking solution PBS with 0.1% v/v Tween-20 (Sigma-Aldrich P9416) and 1% w/v bovine serum albumin (BSA; Jackson Immunoresearch 001-000-162)
- the following sets of primary and secondary antibodies were used: for imaging nuclear speckles, a primary antibody against SC35 (Abeam, abl 1826) was used - a splicing factor commonly used as a marker of nuclear speckles - at 1:200 dilution from stock and a donkey anti-mouse secondary antibody labeled by a Cy5 dye (Jackson Immunoresearch, 715- 175-150) diluted 1:1,000 from stock concentration.
- anti-fibrillarin antibody (Abeam, ab5821), at 1:200 dilution from stock, and a donkey anti-rabbit secondary antibody labeled by an Alexa 657 dye (Jackson Immunoresearch, 711-605-152), diluted 1:1,000 from stock concentration was used.
- the immunofluorescence staining was performed immediately after RNA imaging using the anti- geminin antibody (Abeam, abl95047), at 1:100 dilution from stock, and a donkey anti-rabbit secondary antibody labeled by an Alexa 657 dye (Jackson Immunoresearch, 711-605-152), diluted 1:1,000 from stock concentration.
- Each camera FOV used either 1,000 x 1,000 pixels, with a camera pixel corresponding to 153 nm in each dimension in the imaging plane, or 2048 x 2048 pixels, with a camera pixel corresponding to 108 nm in each dimension in the imaging plane.
- z-stack images of each FOV were acquired in 3 or 4 colors: 647 nm and 750 nm illumination (or 560 nm, 647 nm, and 750) were used to acquire FISH images, 560 nm illumination (or 405 nm illumination) was used to image fiducial beads.
- 647 nm illumination was used to image the DAPI signal
- 647 nm excitation channel was used after RNA or DNA imaging.
- Consecutive z-sections were separated by 85, 100, 150 or 200 nm, covering the entirety of the nuclear volume for all imaged cells. At each z position, images were acquired in all channels before the stage was moved and images were acquired at a rate of -10 Hz.
- the buffer used for cleaving contained 50 mM tris(2-carboxyethyl)phosphine (TCEP; Sigma- Aldrich, C4706) to reduce the disulfide bond connecting fluorophores to readout probes, as well as ImM dye-free common readout probes in 35% formamide to block any unoccupied readout sequences from interfering with the next round of hybridization.
- TCEP tris(2-carboxyethyl)phosphine
- Image analysis Overview of analysis pipeline.
- the image analysis pipeline used in this study was implemented in Python.
- the overall pipeline used the following steps: identify and segment all imaged nuclei, fit 3D-Gaussians to all detected fluorescent spots in imaging channels used for DNA or RNA imaging, as well as for fiducial beads. DNA and RNA spots which did not overlap with identified nuclei were rejected, correct sample drift using the fiducial beads, correct chromatic effects between different color channels, and assign identities to DNA loci and RNA molecules using custom algorithms and software (these are described separately below for DNA and RNA imaging, both for chromosome wide and for genome- wide imaging).
- DAPI images from the first round of imaging were used to identify the volume of individual nuclei and allowed for cell segmentation. This was achieved via a convolutional neural network, built and trained similarly to a previous published work, which took the maximum projection of the DAPI image onto the xy plane as input.
- the number of fitted spots per image that will be retained for decoding was fixed to 125 or fewer in genome-scale imaging by combinatorial FISH ( ⁇ 3-fold greater than the number of distinct loci expected without noise).
- the number of fitted spots per chromosome per image was fixed to 6 or fewer.
- the fitted spots from step 3 were then used for identifying DNA loci and transcription foci and determining their positions, as described in the corresponding sections below. Drift correction.
- Fiducial bead spot fitting was performed in the same way as described above. The set of fiducial bead positions was then compared between rounds of hybridization and a rigid transformation was applied to minimize the sum of square difference of the relative position of beads.
- Bleedthrough and chromatic aberration for multi color imaging were performed by labeling the same set of genomic loci in each imaging channel independently and comparing the signals of the same loci in the different color channels, respectively.
- DAPI signal was first used for rough image-registration across the two sets of images (i.e. chromatin and RNA) via 2D image correlation (all images within each set were aligned to the DAPI image using fiducial beads).
- RNA decoding was performed (see “Identification of transcription foci from fitted RNA spots in sequential imaging” and “Decoding algorithm for fitted RNA spots in combinatorial, genome-scale imaging” below).
- a finer alignment was calculated by assuming that the displacement between nascent RNA localization and their harboring DNA loci should average to zero when considered across all imaged genes and cells in a field of view.
- an additional rigid transformation was calculated to minimize the mean displacement between imaged nascent RNA and their corresponding DNA loci and used this as the final alignment.
- Identification of chromatin loci from fitted DNA spots in sequential imaging Identification and 3D localization of each locus were achieved through the following steps: a list was generated for the drift- and aberration-corrected locations of all fitted spots in each image. Because the spot finding algorithm was allowed to find up to 6 candidates for each chromosome in each sequential image corresponding to a specific color channel of a specific hybridization round (see “Spot fitting for DNA and RNA imaging”), the following additional steps were performed to identify the candidate spots most likely to originate from the imaged chromatin locus. An initial tentative chromatin trace was generated by selecting the brightest spot corresponding to each chromosome copy in each cell in each color channel of each hybridization round.
- the spot For each fitted spot, regardless of whether it was selected for the initial tentative chromatin trace, three quality metrics were calculated: the spot’s brightness above local background, the spot’s distance to the local center of mass, which was calculated from five loci upstream and five downstream along the tentative chromosome trace, and the distance to the center of mass of the entire tentative chromosome trace.
- the three quality metrics described above were combined into a single measure by calculating the combined Fisher p-value for every candidate spot against the distribution of quality metric values for spots included in the tentative chromatin trace (which we will term the “valid distribution”).
- the quality scores of loci that had been included in the re-imaging experiment were first computed to determine displacement error (see “Identification of re imaged loci in sequential imaging” section, below). Then, the distribution of quality scores for spots with low displacement error ( ⁇ 500 nm) between the original was calculated and loci were re-imaged for spots with high displacement error (>500 nm). Finally, the quality score threshold was set such that the fraction of loci in the final chromatin trace (after applying the threshold) expected to be in the high displacement error category is ⁇ 5%. The remaining spots after step 5 were used to determine the final positions of the chromatin loci and trace the chromatin structure.
- RNA imaging rounds Signal from RNA imaging rounds was analyzed using the following procedure: first, the positions of fitted RNA spots for each cell was corrected for chromatic aberration and drift using first a coarse DAPI-based alignment and the brightest RNA spot within a distance of 1000 nm to the corresponding DNA locus was kept. Then, the registration between DNA and RNA imaging was refined based on the displacement between the initial selected RNA localizations (from step 1 and the location of the DNA locus harboring them as described in the “Image registration between DNA and RNA imaging” section above. Lastly, the locations of all the candidate RNA spots, after the fine registration from step 2, were compared to the location of the 50kb DNA locus harboring the gene and the corresponding 5kb DNA transcription start site.
- Identification of re-imaged loci in sequential imaging and estimate of displacement error was performed similarly to the description in the “Identification of chromatin loci from fitted DNA spots in sequential imaging” section, except that the re-imaged loci were used to replace the corresponding subset of loci in the original imaging rounds.
- loci that are far away from both of their genomic neighbors may have a relatively low confidence.
- Decoding algorithm for fitted DNA spots in combinatorial, genome-scale imaging Identification and 3D localization of each locus were achieved through the following steps: first, a list was generated for the drift- and aberration-corrected locations of all identified spots in each bit-image (corresponding a specific color channel in a specific round of imaging). For each detected spot in every bit-image, all spots from other bit- images that were within a set cutoff distance (-150 nm in x, y and z) from its location were found. All such pairs of spots were retained for further analysis, whether or not the barcode produced by a spot pair (based on which round and color channel they appeared in) corresponded to a valid barcode (i.e. a barcode that was assigned to a genomic locus).
- the distribution of spot-pair quality metrics from the invalid barcodes is referred to as the “invalid distribution” and from all valid barcodes as the “valid distribution.”
- the three quality metrics in step 3 were combined into a single measure by calculating the combined Fisher p-value for every candidate spot pair against the “valid distributions.” This can be thought of as the overall quality score of each spot-pair, and was calculated per pair in the following way: for each of the three metrics the fraction of other spot-pairs in the “valid distribution” that had lower quality metric were calculated and these three fractions were multiplied.
- the points between the groups were progressively switched to first maximize the fraction of assigned points in each homolog and then minimize the radius of gyration of each homolog.
- After separating the two homologs their center of mass and the distance of each spot-pair from step 2 to their parent chromosome’s center of mass were calculated.
- the distance to the chromosome center was added as another quality metric in addition to the 3 metrics considered in step 3 and repeated steps 3-6.
- the spot pairs from step 7 were filtered to remove the pairs whose quality scores remained similar to the “invalid distribution.”
- the remaining spot pairs after step 8 were used to determine the final positions of the chromatin loci and trace the chromatin structure.
- RNA bursts Decoding algorithm for fitted RNA spots in combinatorial, genome-scale imaging. Signal from RNA imaging rounds was decoded using the following procedure: first, a list was generated for the drift- and aberration-corrected locations of all identified spots in each round of imaging. For each detected spot in every imaging round, all spots from other rounds that were within a set cutoff distance from its location were found and these spot pairs were retained as candidate RNA bursts if they formed a valid barcode. Then, the location of each of these candidate RNA bursts was then compared to the location of the DNA locus harboring the relevant gene, after initial image registration (based on DAPI images) and drift and aberration correction, and kept if they were within a set threshold distance.
- RNA localization was refined based on the displacement between the initial decoded RNA localizations (from step 3) and the location of the DNA locus harboring them as described in the “Image registration between DNA and RNA imaging” section above.
- locations of all candidate RNA bursts were compared again to the location of the DNA locus harboring the gene to which they decode, this time with the refined image registration. If the nascent RNA localization was within a cutoff distance from its harboring DNA locus at this stage, it was considered as a detected transcriptional burst.
- nuclear bodies from immunofluorescence imaging.
- the location of nuclear bodies was extracted from immunofluorescence signals by applying a threshold to the intensity of the immunofluorescence signals, resulting in a pixelated mask identifying high immunofluorescence signals. This was then treated as a pixelated set of locations “containing” nuclear bodies.
- the position of the nuclear lamina was estimated by generating the minimal 3D convex hull surface (using Python’s SciPy package) surrounding the locations of all decoded chromatin loci in a given cell.
- Spatial distance The spatial distance between any pair of loci was simply calculated as the Euclidean distance between their fitted 3D Gaussian centers, multiplied by the appropriate ratios relating camera pixels and z steps to physical distance.
- the minimal Euclidean distance to all identified nuclear body “locations” or the minimal distance to the surface of the convex hull defining the nuclear lamina was calculated.
- Proximity frequency matrices from imaging To calculate the proximity frequency between any given pair of loci, the number of measured distances between that locus-pair that was smaller than a set cut-off distance was first counted (500 nm in this study, unless otherwise mentioned). This number was then divided by the total number of distances measured for that pair of loci. The cut-off distance was determined by assessing the Pearson correlation between the proximity frequency matrices resulting from a range of cut-off thresholds with the Hi-C contact matrix, as well as the alignment of ensemble structural features, such as TADs and compartments, derived from imaging and Hi-C data for Chr21.
- the spatial distances between each pair of chromatin loci for each cell were calculated.
- the local A/B density ratio was computed in the following way: first, a Gaussian probability density function centered around each A or B locus with a standard deviation of 100 nm (for Chr21 imaging), 125 nm (for Chr2 imaging), or 500 nm (for genomic-scale imaging) was placed. Then, the total A density at the locus was then computed as the sum of this Gaussian probability density function values from all A loci excluding itself in whole-chromosome imaging.
- the total trans A density at the locus were summed from all /ran.s-chromosomal A loci (i.e. all A loci from other chromosomes).
- the total B density was computed in an analogous way.
- the total density of compartment-A loci was divided by the density of compartment-B loci to find the A/B density ratio at the locus.
- the trans A/B density ratio was computed analogously. Insulation score from imaging data. Insulation score has been previously defined for ensemble Hi-C. An analogous definition was used for the imaging results and applied to compute insulation scores of neighboring or non-neighboring domains in individual chromosomes in single cells.
- an intra-domain distance distribution was calculated by considering all distances between each pair of loci within the first domain and all distances between each pair of loci within the second domain.
- An inter domain distance distribution was then calculated by considering all distances between pairs of loci that reside in different domains.
- the insulation score was then defined as the median of all inter-domain distances divided by the median of intra-domain distances. Two highly intermixed domains would have insulation score close to 1, while domains that are just contacting will have an insulation score of ⁇ 2.
- A/B segregation score within chromosomes quantifies the level of spatial separation between A and B loci within a chromosome.
- the A-dense volume was first operationally defined within each chromosome as the 3D space that contains all A loci with A density scores being in the top 2/3 range.
- the B- dense volume was operationally defined in an analogous fashion.
- a purity metric of the A- and B-dense volumes was defined as the fraction of all loci within these volumes being A and B loci, respectively.
- the A/B segregation score was defined as the mean of purity values of the A-dense and the B -dense volumes. This segregation score would be 1 if A and B loci are entirely segregated, and a chromosome with A and B loci completely intermixed would have segregation score around 0.5.
- Hi-C data analysis Hi-C data for IMR-90 cells was procured from and loaded using straw. For identification of A/B compartments in individual chromosomes, established published protocols were followed. For identification of TADs, the method described in the “Normalized insulation score for TAD calling” section was used. For comparison of proximity frequencies derived from the imaging data to Hi-C number of contacts, bins centered around the regions targeted were created and Hi-C data for these bins was procured by summing the number of reads in higher resolution Hi-C data.
- CTCF and Rad21 ChIP-seq data analysis CTCF and Rad21 ChIP-seq data analysis.
- CTCF and Rad21 ChIP-seq data were downloaded from ENCODE dataset and converted to wig format by UCSC Genome Browser Utilities. Read counts for the targeted genomic segment were collected and normalized by input correspondingly. Focal maxima of CTCF or Rad21 ChIP-seq signal enrichment over input along the chromosome were called by standard peak calling algorithm from Scipy.
- a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
- the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
- This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.
- “at least one of A and B” can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one,
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Molecular Biology (AREA)
- Medical Informatics (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Theoretical Computer Science (AREA)
- Evolutionary Biology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Computer Vision & Pattern Recognition (AREA)
- Microbiology (AREA)
- Public Health (AREA)
- Evolutionary Computation (AREA)
- Epidemiology (AREA)
- Databases & Information Systems (AREA)
- Data Mining & Analysis (AREA)
- Crystallography & Structural Chemistry (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Bioethics (AREA)
- Immunology (AREA)
- Software Systems (AREA)
- Artificial Intelligence (AREA)
- Signal Processing (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201962954720P | 2019-12-30 | 2019-12-30 | |
US202063060947P | 2020-08-04 | 2020-08-04 | |
PCT/US2020/065797 WO2021138078A1 (en) | 2019-12-30 | 2020-12-18 | Genome-scale imaging of the 3d organization and transcriptional activity of chromatin |
Publications (2)
Publication Number | Publication Date |
---|---|
EP4085150A1 true EP4085150A1 (en) | 2022-11-09 |
EP4085150A4 EP4085150A4 (en) | 2024-04-17 |
Family
ID=76687247
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP20910372.0A Pending EP4085150A4 (en) | 2019-12-30 | 2020-12-18 | Genome-scale imaging of the 3d organization and transcriptional activity of chromatin |
Country Status (7)
Country | Link |
---|---|
US (1) | US20230348958A1 (en) |
EP (1) | EP4085150A4 (en) |
JP (1) | JP2023509010A (en) |
CN (1) | CN115023502A (en) |
AU (1) | AU2020418497A1 (en) |
CA (1) | CA3161593A1 (en) |
WO (1) | WO2021138078A1 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10240146B2 (en) | 2014-07-30 | 2019-03-26 | President And Fellows Of Harvard College | Probe library construction |
WO2023097244A1 (en) * | 2021-11-24 | 2023-06-01 | Yale University | Methods of determining chromatin alterations |
US11834714B2 (en) | 2021-12-20 | 2023-12-05 | Enumerix, Inc. | Detection and digital quantitation of multiple targets |
WO2023122041A1 (en) * | 2021-12-20 | 2023-06-29 | Enumerix, Inc. | Detection and digital quantitation of multiple targets |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10240146B2 (en) * | 2014-07-30 | 2019-03-26 | President And Fellows Of Harvard College | Probe library construction |
CA2968376C (en) * | 2014-11-21 | 2020-06-23 | Nanostring Technologies, Inc. | Enzyme- and amplification-free sequencing |
US20190264270A1 (en) * | 2016-11-08 | 2019-08-29 | President And Fellows Of Harvard College | Matrix imprinting and clearing |
-
2020
- 2020-12-18 WO PCT/US2020/065797 patent/WO2021138078A1/en unknown
- 2020-12-18 CA CA3161593A patent/CA3161593A1/en active Pending
- 2020-12-18 AU AU2020418497A patent/AU2020418497A1/en active Pending
- 2020-12-18 EP EP20910372.0A patent/EP4085150A4/en active Pending
- 2020-12-18 JP JP2022539655A patent/JP2023509010A/en active Pending
- 2020-12-18 CN CN202080087114.8A patent/CN115023502A/en active Pending
- 2020-12-18 US US17/770,943 patent/US20230348958A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
JP2023509010A (en) | 2023-03-06 |
WO2021138078A1 (en) | 2021-07-08 |
AU2020418497A1 (en) | 2022-04-28 |
CA3161593A1 (en) | 2021-07-08 |
CN115023502A (en) | 2022-09-06 |
EP4085150A4 (en) | 2024-04-17 |
US20230348958A1 (en) | 2023-11-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Xia et al. | Multiplexed detection of RNA using MERFISH and branched DNA amplification | |
Su et al. | Genome-scale imaging of the 3D organization and transcriptional activity of chromatin | |
WO2021138078A1 (en) | Genome-scale imaging of the 3d organization and transcriptional activity of chromatin | |
US20230032082A1 (en) | Spatial barcoding | |
EP3538867B1 (en) | Multiplexed imaging using merfish, expansion microscopy, and related technologies | |
Kishi et al. | SABER amplifies FISH: enhanced multiplexed imaging of RNA and DNA in cells and tissues | |
Moffitt et al. | RNA imaging with multiplexed error-robust fluorescence in situ hybridization (MERFISH) | |
US20240112755A1 (en) | Matrix imprinting and clearing | |
US20240175081A1 (en) | Systems and methods for high-throughput image-based screening | |
Emanuel et al. | High-throughput, image-based screening of pooled genetic-variant libraries | |
CN107208144B (en) | Enzyme-free and amplification-free sequencing | |
JP2022537048A (en) | Signal encoding methods for analytes in samples | |
US10851411B2 (en) | Molecular identification with subnanometer localization accuracy | |
Dardani et al. | ClampFISH 2.0 enables rapid, scalable amplified RNA detection in situ | |
Cardozo Gizzi et al. | Direct and simultaneous observation of transcription and chromosome architecture in single cells with Hi-M | |
US20230212556A1 (en) | Systems and methods for associating single cell imaging with rna transcriptomics | |
US20210171939A1 (en) | Sample processing barcoded bead composition, method, manufacturing, and system | |
Kinrot | Exploring Single-Cell Chromatin Organization with Multiplexed DNA-FISH: Towards an Imaging Platform for Single-Cell Multi-Omics | |
Moffitt et al. | RNA Imaging with Multiplexed Error Robust Fluorescence in situ Hybridization | |
WO2023172915A1 (en) | In situ code design methods for minimizing optical crowding | |
Wadsworth et al. | FISHing on a Budget | |
Situ | RNA Imaging with Multiplexed |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE |
|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE |
|
17P | Request for examination filed |
Effective date: 20220512 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
DAV | Request for validation of the european patent (deleted) | ||
DAX | Request for extension of the european patent (deleted) | ||
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R079 Free format text: PREVIOUS MAIN CLASS: C12Q0001680000 Ipc: G16B0015100000 |
|
A4 | Supplementary search report drawn up and despatched |
Effective date: 20240319 |
|
RIC1 | Information provided on ipc code assigned before grant |
Ipc: C40B 70/00 20060101ALI20240313BHEP Ipc: C12Q 1/6841 20180101ALI20240313BHEP Ipc: C12Q 1/6837 20180101ALI20240313BHEP Ipc: C12Q 1/6816 20180101ALI20240313BHEP Ipc: C12Q 1/68 20180101ALI20240313BHEP Ipc: G16B 40/10 20190101ALI20240313BHEP Ipc: G16B 15/10 20190101AFI20240313BHEP |